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CN102939380A - Methods for cloning and manipulating genomes - Google Patents

Methods for cloning and manipulating genomes Download PDF

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CN102939380A
CN102939380A CN2010800666205A CN201080066620A CN102939380A CN 102939380 A CN102939380 A CN 102939380A CN 2010800666205 A CN2010800666205 A CN 2010800666205A CN 201080066620 A CN201080066620 A CN 201080066620A CN 102939380 A CN102939380 A CN 102939380A
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cell
gene group
donor
host
nucleic acid
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G·A·本德斯
J·I·格拉斯
C·A·胡赤森
C·拉蒂格
S·瓦希
M·A·奥吉拉
H·O·史密斯
C·E·玛丽曼
V·N·诺斯克
R-Y·闯
D·G·吉布森
J·C·文特尔
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Viridos Inc
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Synthetic Genomics Inc
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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Abstract

Compositions and methods are disclosed herein for cloning a synthetic or a semi-synthetic donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

Description

For cloning and operate genomic method
Being incorporated to of sequence table
The application comprises the quoting of aminoacid sequence and/or nucleotide sequence, and itself and the application submit to via EFS-Web simultaneously, and the sequence list text presents is " SGI1270-2WO_ST25.txt ", and file size 106,496 bytes created on March 19th, 2010.Above-mentioned sequence table is incorporated to its integral body by reference thus according to 37 C.F.R. § 1.52 (e) (5).
Background technology
The biology that use has high genetic system allows the nucleotide sequence of operation separation in the host as the host of the nucleic acid molecule separated from many species.But, owing to transferable, entering to have the restriction of the genetic species of easy processing such as the nucleic acid molecule size in yeast, in foreign host, by clone and the ability of modifying karyomit(e) and genome manipulation biology, be restricted.
For example, although (, DNA) be transferred to host cell, the nucleic acid of cloning by ordinary method generally includes only minority gene to larger nucleic acid.For example, 16kb mouse mitochondrial genome be cloned into intestinal bacteria (Itaya etc., NatMethod 5,41 (2008); Yoon and Koob, Nucleic Acids Res 31,1407 (2003)), Bacillus subtillis (Bacillus subtilis) (Itaya etc., Nat Method 5,41 (2008); Yoon and Koob, Nucleic Acids Res 31,1407 (2003)) and in yeast (Wheeler etc., Gene 198,203 (1997)).139kb DCIPThe chloroplast of maize genome has been cloned into yeast (Gupta and Hoo, Plant MoIBiol 17, in 361 (1991), Bacillus subtillis (B.subtilis) (Itaya etc. have been cloned into 135kb rice chloroplast genome, NatMethod 5,41 (2008)) in.1.8Mb hemophilus influenzae (Haemophilus influenzae) genomic about 10% is cloned into intestinal bacteria (Smailus etc., Syst Synth Biol as additive type element (episomal element); 1,139 (2007)) in.3.5Mb synechocystis (Synechocystis) PCC6803 genome is inserted into genomic three the discontinuous zones of Bacillus subtillis, except two ribosome-RNA(rRNA) operons (Itaya etc., PNAS USA 102,15971 (2005)).Fully synthetic 0.6Mb mycoplasma genitalium (Mycoplasma genitalium) genome be used as annular yeast centromeric plasmid (YCp) be fitted into yeast (Gibson etc., Science 319,1215 (2008); Gibson etc., PNAS USA, 105 (51): 20404-9 (2008)).
U.S. Patent number 6,670,154 have described by merging bacterium and yeast---and make the genome linearizing of modifying, make the bacterial genomes of modifying be converted into the chromosomal method of artificial yeast.U.S. Patent Application Publication No. 2005/0019924 has been described for introducing eukaryotic cell using Prokaryotic genome as ring molecule and being converted into nucleic acid and the method for artificial chromosome.WO 02/057437 has described and has comprised the genomic yac vector of cytomegalovirus (CMV).U.S. Patent number 7,083,971 have described recombination method and the system for clone, operation and conveying large nucleic acids fragment.U.S. Patent Application Publication No. 2005/0003511 and Bradshaw etc., Nucleic Acids Research, 23,4850-56 (1995) has described yeast for clone large DNA zone by homologous recombination-bacterium shuttle vectors.
But, disclosed clone and working method are subject to the transferable restriction that enters the donor nucleic acid size in host cell, and operation can not be provided and/or shift the nucleic acid molecule of breeding in host cell to be back in the recipient cell relevant to donor, they can not solve the different cell types of clone's use and the inconsistent problem between external nucleic acid.Need other method for cloning large nucleic acid such as karyomit(e) or genome enter alternative heterologous host, are back to and receptor biological like the donor biophase for the sequence at alternate host operation large nucleic acids with for the genome of jump operation, same genus biological (for example, from the protokaryon to the eukaryotic cell and return) for example.
So far, not of the same race or do not belong to nucleic acid that between biology, transfer is large together and do not overcome yet such as karyomit(e) and genomic obstacle.For example, between planting, the transfer of nucleic acid can have toxicity to host, donor and/or recipient cell.Not of the same race, belong to or group's biology neutralization from the prokaryotic cell prokaryocyte to the eukaryotic cell and the nucleic acid returned operation and propagation also can cause the unstable of nucleic acid and suppress their activation, such as the expression of the gene from nucleic acid.
Summary of the invention
This paper provides for donor nucleic acid is shifted to (clone) and has entered host cell; For example, for example, for operation (, modifying) donor nucleic acid, in host cell; For the donor nucleic acid of modification being transferred to method, nucleic acid and the system of recipient cell.The method provided and other compositions the nucleic acid of whole biology branch shift and operation in be useful, such as at eukaryotic host cell operation protokaryon nucleic acid and transfer nucleic acid, returning to former nuclear receptor.
Method can be used for by be transferred to there is strong well-characterized genetic system such as in yeast, operation has the donor nucleic acid of biology of weak genetic system.Therefore, the method, nucleic acid and system can be used for modifying the large nucleic acids that the nucleic acid of unmanageable biology operation and transformation comprise genome, for example produce synthetic gene group and cell, such as non-existent cell and genome in laboratory or nature before.The method provided can be used for the clone, modifies and shift the nucleic acid and the genome that are greater than 300 kilobase (kb), such as genome, comprises full genome and at least minimal genome and cell, virus and organelle gene group.Thereby the donor gene group can be modified to produce the donor gene group of modification in host cell, it gives natural donor gene group otherwise one or more phenotypes that can not show.While being difficult to produce in the donor gene group of these modifications is having the initiating cell type of donor gene group, or work as the synthetic gene group and can in host cell, assemble and modify fast, when the genome that then makes to modify shifts in the cell type that returns to original expectation for generation of interested phenotype or product, the method is especially favourable.
The composition of identifying in the application and describing and method allow nucleic acid molecule and genome are transferred to the novel method of host cell from unmanageable donorcells, in this host cell, thereby they can be modified to change genotype and be changed phenotype, to change nucleic acid molecule or genome.The genome of modifying can be used the hereditary machine of host cell to modify in one or more modes.The method provided also is provided for separating from host cell nucleic acid molecule or the genome of modifying.The nucleic acid molecule of the modification separated or genome can methylate by first external rear body interior (ex vivo).Recipient cell can be processed and be transferred to cell with the nucleic acid molecule by modification or genome by method described herein.Then, can further the nucleic acid molecule of modification or genome be transferred to recipient cell, thereby change nucleic acid molecule or the genomic phenotype of recipient cell phenotype for modifying.
This paper provides for cloning the method for donor gene group, and it comprises: as one or more fragment, from donorcells, obtain donor gene group or synthetic donor gene group; With donor gene group and host's carrier are introduced to the heterologous host cell, wherein optionally the donor gene group was connected before introducing host cell with host's carrier, thereby generate the host cell that comprises the donor gene group, described donor gene group comprises host's carrier, and further wherein the donor gene group is basically complete cell, virus or organelle gene group, it is at least minimal genome, and length is greater than about 300 kb.In one embodiment, the donor gene group is the genome of full cell, virus or organoid basically.
In method described herein, donor gene group and host's carrier can or be introduced host cell in succession simultaneously.If donor gene group and host's carrier are introduced host cell in succession, can introduce with random order.Therefore, in one embodiment, the donor gene group can be introduced host cell, introduces subsequently host's carrier.Alternatively, host's carrier can be introduced host cell, introduces subsequently the donor gene group.In another embodiment, by host's carrier being transformed into to the donorcells that comprises the donor gene group, host's carrier is connected with the donor gene group, then introduces host cell.
The donor gene group can be unit molecule.In one embodiment, the nucleic acid molecule that comprises donor gene group and host's carrier can be used as ring-type kinetochore plasmid and exists.Alternatively, the donor gene group can be used as the existence of overlapping DNA fragment.Before introducing host cell, can be by the linearizing of donor gene group or fracture.
Some embodiment of the method provided also comprises recovery donor gene group and host's carrier from host cell.
In other embodiments, the method also comprises donor gene group introducing recipient cell.
In other embodiments, the method provided also comprises degraded or removes the genome of recipient cell.
The donor gene group that this paper considers includes but not limited to fungal gene group, Archimycetes genome, cyanobacteria genome, algae genome, viral genome, phage genome, organelle gene group, Mitochondrial Genome Overview, chloroplast gene group (for example, DCIPThe chloroplast of maize genome or rice chloroplast genome), organelle gene group or synthetic gene group.
The host cell that this paper considers is eukaryotic cell or prokaryotic cell prokaryocyte.Host cell includes but not limited to, bacterial cell, fungal cell, insect cell, vegetable cell or alga cells.Host cell also comprises yeast cell.
Host's carrier described herein can be the kinetochore plasmid.A kind of preferred embodiment in, host's carrier is that yeast centromeric plasmid and host cell are yeast cell.
Host's carrier described herein is the carrier that can be used for homologous recombination.
Any method described herein can further be included in host cell modifies the donor gene group.
In addition, any method described herein can further comprise the donor gene group that comprises host's carrier from the host cell recovery.Optionally, host's carrier can be removed from the donor gene group.On the one hand, method also comprises the donor gene group introducing recipient cell that will reclaim.
Method described herein can further comprise degraded or the native gene group of removing recipient cell.When method comprises the first situation of donor gene group introducing recipient cell, the native gene group of recipient cell is the natural gene group.When carrying out many wheel modifications (see, for example, Fig. 1 and 16), the native gene group of recipient cell can be the previous genome of modifying or synthetic genome.Therefore, in one embodiment, the method provided also comprises that (repeat, mode iterative) is modified the donor gene group with iteration.
Can be before introducing recipient cell, by the one or more Nucleotide in the donor gene group that methylates, the donor gene group that methylates and reclaim.
Recipient cell can be, for example bacterial cell, yeast cell, fungal cell, insect cell, vegetable cell or alga cells.On the one hand, before introduce the donor gene group, the restriction enzyme function of recipient cell be non-existent, be removed or inactivation.For example, can in recipient cell, suddenly change restricted modifying enzyme so that its inactivation.
In a preferred embodiment, the donor gene group comes from prokaryotic cell prokaryocyte (nature or synthetic) and is cloned into eukaryotic cell, and optionally it can be modified there, and then reclaims and introduce and return in eukaryotic cell.In some preferred implementation, the donor gene group comes from bacterial cell (nature or synthetic) and is cloned into yeast cell, and optionally it can be modified there, and then reclaims and introduce and return in bacterial cell.
Method provided herein also comprises introduces host cell by the second donor gene group, and wherein the second donor gene group is different from the first donor gene group, thereby produces the host cell that comprises two different donor gene groups.Can by the host cell that makes to comprise the first donor gene group, with the second host cell mating that comprises the second donor gene group, (combination mate), be introduced the second donor gene group.But the donor gene group of recovery is introduced on the recipient cell phenotype recipient cell is changed into and the corresponding phenotype of donor gene group that is incorporated to any modification.
This paper provides the method for the manufacture of cell, described cell display is by the phenotype of donor gene group coding, described method comprises: (a) donor gene group and the host's carrier that is adapted at clone's donor gene group in host cell are introduced to host cell, obtain like this product that comprises the donor gene group, described donor gene group comprises host's carrier; (b) be recovered in from host cell the product that comprises the donor gene group obtained step (a), described donor gene group comprises host's carrier; (c), the product of (b) is introduced to recipient cell under certain condition, so that recipient cell is showed the phenotype by this product coding; (d) reclaim the cell obtained from step (c); Wherein the donor gene group is basically complete cell, virus or organelle gene group, it is at least minimal genome, and the minority component that length is greater than about 300 kb and comprises nucleic acid substances, described minority component shows that for recipient cell the phenotype by the donor gene group coding is essential.On the one hand, method also is included in the donor gene group of modifying (a) in host cell.On the other hand, method also comprises degraded or the native gene group of removing recipient cell.On the other hand, method also is included in the donor gene group of modifying (a) in host cell the native gene group of degrading or removing recipient cell.In some embodiments, these methods can be automatizations.
This paper provides the cell of showing the expectation phenotype, described expectation phenotype by the donor gene group coding and otherwise can be by cell display, wherein cell is produced by method described herein.
This paper also provides the cell that comprises the donor gene group and show the expectation phenotype, described expectation phenotype by the donor gene group coding and otherwise can be by cell display, wherein the donor gene group comprises the foreign gene group nucleic acid substances that is greater than 300kb and the genome component of essential minimum for cell display expectation phenotype.The expectation phenotype can comprise the natural expression product of the non-initial cell of generation, or modifies (for example, selective expression or regulated expression) or the existing expression product of incremental adjustments.
The method provided also is included in a plurality of genomes of clone in a plurality of host cells.A plurality of genomes can be the genome variants.In one embodiment, a plurality of genomes are introduced to host cell and comprise host's carrier and a plurality of variant overlapping fragments introducing host cell, thus the combinatorial library of generation variant gene group.
On the one hand, reclaim the donor gene group from host cell after, the method provided comprises introduces recipient cell and recipient cell than host cell higher degree ground, to support the genetic expression from the donor gene group by the donor gene group.
On the one hand, the method provided is included in host cell modifies the donor gene group; Be included in the donor gene group and induce one or more replacements, one or more disappearance, one or more insertion, one or more rearrangement, one or more restructuring, one or more homologous recombination or its combination with modification donor gene group.
On the other hand, method comprises modification donor gene group; And with the donor gene group before modifying, compare, the modification of donor gene group realizes or improves the character of donor gene group.
The method provided also comprises that transfer enters recipient cell, wherein can be in the situation that exist polyoxyethylene glycol (PEG) to be shifted.The PEG of spendable all size includes but not limited in the method, and scope is from the size of PEG 4,000 to PEG 20,000.In one embodiment, size is PEG 8,000.Can use various PEG concentration in disclosed method, such as, for example from about 1% to about 20%.In one embodiment, use PEG with about 5% concentration.
This paper provides the carrier for full genomic modification, and it comprises it being at least the Prokaryotic genome of minimal genome; The protokaryon replication origin; The protokaryon selective marker; Transposase and inverted repeats; One or more can be supported in the nucleotide sequence that separates and copy in eukaryotic cell; With the eucaryon selective marker.In the one side of the method provided, eukaryotic cell is yeast cell.Prokaryotic genome, protokaryon replication origin and selective marker can be bacteriums.In one embodiment, be supported in eukaryotic cell the nucleic acid that separates and copy and comprise one or more CEN nucleic acid and ARS nucleic acid.In another embodiment, the length of Prokaryotic genome comprises at least at 300kb or about 300kb.In another embodiment, carrier is stable in eucaryon and prokaryotic cell prokaryocyte.This paper provides the combinatorial library that comprises a plurality of carriers, and wherein Prokaryotic genome can be the genome variant.
This paper provides the isolated cell that comprises external source donor gene group, synthetic cell or the reconstitution cell by the preparation of any method described herein.
This paper provides the yeast nucleic acid construct for the target region of seamless modification (seamless modification) target nucleic acid, it comprises: the first homology part, and it comprises along target nucleic acid length in the upstream of target region or the homologue of a part of target nucleic acid in downstream; Under inducible promoter is controlled, the nucleic acid of coding nucleic acid restriction endonuclease; The nucleotide sequence of endonuclease identification; The yeast selective marker; The second homology part, the homologue that it comprises target region 5 ' part; With the 3rd homology part, the homologue of the 3 ' part that it comprises target region.In one embodiment, the second and the 3rd homology part is at the first homology part, the nucleic acid of coding nucleic acid restriction endonuclease and the flank of yeast selective marker.Endonuclease recognition site can be close to the second or the 3rd homology part, and can be with respect to the first homology part the relative end at construction.With homology in target nucleic acid, partly compare, second and one of the 3rd homology zone or both comprise one or more replacements, one or more disappearance, one or more insertion, one or more rearrangement, one or more restructuring, one or more homologous recombination or one or more their combination.
This paper provides the method for modifying in the seamless introducing of target nucleic acid molecule, it comprises: mutagenesis construct and host's carrier are introduced to host cell, whereby host's carrier in host cell with mutagenesis construct restructuring, wherein the mutagenesis construct is included in 5 ' the first homology part partly of the target nucleic acid molecule of this modifications upstream; Gene and the selective marker of endonuclease recognition site, promotor, coding nucleic acid restriction endonuclease; The second homology repeating part with genome sequence homology in target gene seat upstream; The 3rd homology part with 3 ' part of the target region of modifying downstream at this; Incubation cell under certain condition, between the first homology part and upstream or downstream part, recombinated whereby, a thereby seamless part of removing construct, it promotes one or more double-strand breaks cuttings comprise the target site of construct in nucleic acid molecule near, will modify whereby seamless introducing target nucleic acid molecule.
The processing that promotes double-strand break to cut can comprise the expression of endonuclease, and the target nucleic acid molecule that described endonuclease comprises this construct in the cutting of recognition site place, produce double-strand break.On the one hand, the method provided also comprises is selected step, selects whereby the cell that wherein the yeast selective marker has been removed from target nucleic acid.
The method provided comprises and is transferred to recipient cell, is wherein shifted through the following steps: in the situation that agarose exists, separate the donor gene group; In the situation that there is incubation donor gene group in methyltransgerase, donor gene group whereby methylates; Agarose is melted; With by donor gene group incubation together with recipient cell.With incubation together with methyltransgerase can be together with cell crude extract incubation.
The method provided can further be included in together with formyl transferase and, after incubation, in the situation that there is incubation donor gene group in proteolytic enzyme, thereby remove deproteinize.
Typically, the donor gene group that this paper considers and the genome of modification are large nucleic acid.In one embodiment, the length of donor gene group is at least or is approximately or be greater than about 100kb, about 150kb, about 200kb, about 250kb, about 300kb, about 350kb, about 400kb, about 450kb, about 500kb, about 550kb, about about 600kb of 600kb, about 650kb, about 700kb, about 750kb, about 800kb, about 850kb, about 900kb, about 1 megabase (MB), about 1.1MB, about 1.2MB, about 1.3MB, about 1.4MB, about 1.5MB, about 1.6MB, about 1.7MB, about 1.8MB, about 1.9MB, about 2MB, about 2.5MB, about 3MB, about 3.5MB, about 4MB, about 4.5MB or larger, or any numerical value between them.
The accompanying drawing summary
Fig. 1 illustrates numerous embodiments, and wherein donor gene group and host's carrier can (by transforming or cotransformation) be introduced host cell.Can in host cell, carry out one or more genomic modifications.Then, the genome of separable modification be transferred to recipient cell.
Fig. 2 A-2C illustrates in the genomic three kinds of methods of yeast cloning bacteria.(A) in order in conversion, to pass through afterwards Yeast proliferation, can host's carrier be incorporated to bacterial genomes by transform bacteria; The genome of restructuring and host's carrier can be separated and for the transformed yeast host cell.Alternatively, (B) full genome and optionally linearizing host carrier can be dissolved yeast host cell by corotation, pass through there the homologous recombination genome, yeast host cell is in conjunction with carrier and bacterial genomes.In another program (C), bacterial genomes can be cloned by assembling a plurality of overlapping fragmentses and dissolving yeast host cell with host's carrier corotation, wherein by homologous recombination in yeast host cell, by bacterial genomes fragment and the combination of host's carrier.
Fig. 3 A-3F illustrates the method for using Fig. 2 A and inserts at the genomic yeast vector in each of mycoplasma genitalium, mycoplasma mycoides LC and mycoplasma pneumoniae.Fig. 3 A, 3B and 3E illustrate two shuttle vectorss that use in experiment.Fig. 3 C, 3D and 3F illustrate the position that carrier inserts in each genome.The mycoplasma mark is the spiralin promotor, tetM and lacZ; The yeast vector feature is CEN, ARS and HIS3; The escherichia coli plasmid skeleton is amicillin resistance (ampR) and pUC19 initial point (ori); The BAC sequence is BAC; With the transposon element be the outer inverted repeat (outer invented repeat) of IS256 (IR), inverted repeat (inner invented repeat) is (IR) and transposase (tnp) in IS256.
Fig. 4 A-4B shows the analysis of the full genomic clone of mycoplasma that comprises the yeast vector sequence.Fig. 4 A provides the genomic collection of illustrative plates of mycoplasma genitalium c116-2.The on position of yeast vector is labeled.Lines indicating positions and the size of numeral indication pcr amplification.Restricted fragment is numbered and their size is provided in legend; Corresponding to restricted fragment 1, BssHII digests corresponding to restricted fragment 2-6 in EagI digestion.Fig. 4 B provides mycoplasma pneumoniae genomic collection of illustrative plates.The on position of yeast vector is labeled.Lines indicating positions and the size of numeral indication pcr amplification.Restricted fragment is numbered with their size and is provided in legend; NotI digestion corresponding to restricted fragment 1-4 SbfI corresponding to restricted fragment 5 and 6.
Fig. 5 provides the genomic collection of illustrative plates of mycoplasma mycoides LC cll.l.Arrow represents IS 1296 elements.Lines indicating positions and the size of numeral indication pcr amplification.
Fig. 6 A-D illustrates and uses homologous recombination to insert yeast vector as four kinds of optional method targets.In the situation that there is double-strand break and there is no double-strand break, attempt yeast vector in insertion point and insert.Fig. 6 A: complete genome and linear carrier.Fig. 6 B: overlapping genes group fragment and with the carrier of the inside homology of one of fragment.Fig. 6 C: at the genome of integrating target spot and the cutting of linear carrier place.Fig. 6 D: the overlapping genes group and with the carrier segments of two fragment homologies.
Fig. 7 A-7C shows that crude extract protection donor plasmid DNA avoids host's restriction modification system and affects and increase transformation efficiency, but the suppressor gene group shifts.Fig. 7 A illustrates by the step process agarose inserted block that methylates (Agarose plug, agarose plug) or untreated result.Fig. 7 B is presented at and lacks natural gene group DNA (demonstration allows to be transferred to recipient cell) displaying point-like (punctate) pattern (right figure) of processing in the crude extract situation, and in the situation that crude extract exists the native gene group DNA (show and suppress to shift) processed to form large aggregate (left side two figure).Fig. 7 C be presented at the agarose inserted block in after the genomic dna incubation, process and remove crude extract and recovered the dot pattern that the untreated genomic dna of initial use is observed by Proteinase K.
Fig. 8 shows spendable three kinds of optional full genome transfer methods.The first method (1), comprise the agarose inserted block (for example, with β-gelase (melting step)) that digestion comprises genomic dna, then directly is transferred to recipient cell.The second method (2) is identical with the first method, except recipient cell by the restriction enzyme allele of modifying to suddenly change (Δ RE).In third method (3), genome DNA sample is methylated in vitro and is carried out Deproteinization step (processing with Proteinase K), then melts step (β-gelase digestion) and is transferred to recipient cell.
Fig. 9 A-9C illustrates the problem of conventional modifying method with regard to non-specific disappearance or rearrangement aspect.The circle that CEN6=comprises in structure.Fig. 9 A illustrates wild-type fragment is introduced to the yeast that carries the mycoplasma genitalium with URA3 insertion, then on the SD-HIS flat board that comprises FOA, select, obtain the selection of (Pl (restructuring between wild-type fragment and genome) and P2 (restructuring in genome between tumor-necrosis factor glycoproteins), as shown in Fig. 9 B) of two dissimilar recombination event.These events are carried generation in the cell that is illustrated in optional product in Fig. 9 C.
Figure 10 A-10D illustrates optional seamless modifying method.Figure 10 A schematic illustrations the generation of diletto perfetto mutagenesis-cartridge: use the lithium acetate integration transformation, this box introduced and comprised the genomic yeast strain of mycoplasma genitalium.Use diagnostic primers Seq-F and Seq-R (as shown in little single arrow of insertion point both wings), by independent Ura +Transformant is selected by PCR and is analyzed.Generation comprise the URA3 mark and with just at the fusion product of the homeologous 358bp fragment (" repetition " fragment) (being labeled as the large arrow of " repetition ") of target gene seat upstream.For generating final mutagenesis-cartridge (Figure 10 B), fusion product is increased by PCR-again: the homologue of the target region 3 ' part of homologue (upstream of single base deletion), URA3 mark, repetition box and 50 bp of the target region 5 ' part that the box obtained comprises part in the following order: 50bp.With this direction design box, once, in order to be transformed into yeast host cell, with 450 base pair target regions (passing through HR) in genomic CDS 139 locus of this box displacement mycoplasma genitalium, will in genome, produce the zone (being labeled as the large arrow of " repetition ") that comprises two tandem repetitive sequences by the flank in the URA3 selective marker.Figure 10 C: by merging (GAL1/I-SceI)-URA3 fusion product and 358bp " repetition " fragment that is positioned at target gene seat upstream, generate TREC (series connection with endonuclease cutting repeats) mutagenesis construct.Target region 3 ' the homologue (downstream of the single base deletion be corrected) partly of the homologue (upstream of single base deletion) of target region that gained TREC box comprises part in the following order: 50bp 5 ' part, CORE box (gene and URA3 mark by 18bp I-SceI recognition site, GAL1 promotor, coding I-SceI endonuclease form), " repetitions " (with just in the 358bp part of the genome sequence homology of target wild type gene seat upstream) and 50bp.The homologue (downstream of single base deletion) of the target region 3 ' part of homologue (upstream of single base deletion), a LoxP site (LoxP-RE), GAL1 promotor, Cre recombinase gene ORF, URA3 mark, the 2nd LoxP site (LoxP-LE) and 50 bp of the target region 5 ' part that gained LoxP-RE-GAL1-Cre-URA3-LoxP-LE mutagenesis-cartridge (Figure 10 D) comprises part in the following order: 50bp.
Figure 11 illustrates the generation of III type restriction enzyme disappearance.In order to manufacture mycoplasma mycoides LC III type restriction enzyme allele (typeIIIres) disappearance in yeast, by making two PCR product C ORE and tandem repetitive sequence (TRS), merge, build linear DNA fragment---knock out box (KOC).Then, this box is transformed in the yeast W303a bacterial strain that comprises mycoplasma mycoides LC genome-YCp with the displacement of the 50-bp homologous sequence by target site (Δ typeMres::URA3) RIII type ORF.Semi-lactosi is induced and is made I-SceI endonuclease expression of enzymes, and it cuts 18-bpI-SceI site (asterisk), produces double-strand break, and this is conducive to two homologous recombination (red arrow) between tandem repetitive sequence.Restructuring between TRS produces the seamless disappearance (seamless deletion) (Δ typeIIIR) of typeIIIres gene.
Figure 12 A-12B illustrates in the genomic MG259 locus of synthetic mycoplasma genitalium place's through engineering approaches (transformation) point mutation.Figure 12 A illustrates the scheme of proofreading and correct by the sudden change of two serial homology restructuring.Article two, primer (arrow) Seq-F and Seq-R separate 0.4kb in the MG259 locus, and the insertion of 1.1kb URA3 mark causes producing 1.3kb PCR DNA fragmentation.Figure 12 B illustrates the possibility that the URA3 mark is lost from mycoplasma genitalium YAC.The 5-FOA resistance clone can be derived from wild-type DNA fragmentation displacement URA3 mark (R1) or be derived from the restructuring (R2) between tumor-necrosis factor glycoproteins.The size of tumor-necrosis factor glycoproteins and position are schematic.
Figure 13 illustrates the outline of example T REC method.Target region is replaced with mutagenesis-cartridge, and described mutagenesis-cartridge forms with the DNA fragmentation (arrow shown in) identical with the target site upstream by knocking out CORE (the I-SceI recognition site of l8-bp, the I-SceI gene and URA3 gene that controlled by the GAL1 promotor).Displacement generates the tandem repetitive sequence that comprises CORE.Semi-lactosi is induced the expression of I-SceI, and it produces double-strand break (DSB) in the I-SceI site.DSB is conducive to homologous recombination in the molecule between tumor-necrosis factor glycoproteins, causes the excision of CORE.
Figure 14 A and 14B illustrate respectively and to repeat (pop-out) method of giving prominence to (Figure 14 B) by delitto perfetto method (fixed-point mutation method) (Figure 14 A) or series connection and transform identical locus and produce point mutation or two kinds of additive methods of 450bp disappearance.
Figure 15 illustrates the exemplary generation of final mutagenesis-cartridge construct.
Figure 16 illustrates the donor gene group is moved into to host cell, transforms it, and shifts and make it install back acceptor by genome.In illustrative methods, after the clone has the bacterial genomes of yeast vector in yeast, use all technology of yeast genetic method to produce any combination of inserting, lacking, resetting or modify in bacterial genomes.This modifying gene group is followed separated and is transferred to recipient cell to generate the bacterium of transformation.Before being transferred back to recipient cell, it may be necessary that donor dna is methylated, and to protect it, avoids recipient cell restriction system (one or mores') impact.Can start to repeat from the new genome of new transformation this circulation (dotted arrow).
The regional nucleotide sequence of seamless disappearance that Figure 17 provides YCpMmyc1.1-Δ typeIIIres mycoplasma mycoides genome to shift body, confirmation III type restriction gene is removed by design." typeIIImod " zone of the corresponding gene mapping Figure 19 of italic sequence text; UnderscoreCorresponding " typeIIIres " zone of sequence text; With corresponding " IGR " zone of bold text.Overlapping due between typeIIImod and typeIIIres gene, after disappearance, small portion typeIIIres gene still exists.The initiator codon of typeIIIres gene and terminator codon are underlined enlarge fonts; The terminator codon of typeIIImod gene is illustrated as and underlines, the enlarge font of italic, black matrix.Figure 17 discloses SEQ ID NO:195.
Figure 18 A-18C is presented in yeast between proliferation period, the genomic stability of mycoplasma mycoides YCpMmycl.l.Figure 18 A provides the genomic schematic diagram of YCpMmycl.l.Shown the position of integrating YCp.9 independent primer pairs that use in pcr amplification their approximate position in genome show and corresponding amplicon numbering in Figure 18 B.Figure 18 B, detect in yeast the genomic stability of mycoplasma mycoides between proliferation period by two kinds of methods.In first, the yeast culture bed board that comprises genomic clone was in upper two day of solid synthetic medium that lacks Histidine, and then independent colony pieced together to (patch) to new flat board.In second, the yeast culture that comprises genomic clone grows to saturated, is diluted to 1/100 part and regrow to saturated.Culture was followed bed board in upper two day of solid synthetic medium that lacks Histidine and then independent colony is pieced together on new flat board.In two kinds of methods, isolation of genomic DNA also is used as template in the multiplex PCR amplification, and 9 independent primer pairs that show in figure A are used in described amplification.By gel electrophoresis analysis gained amplicon.Each primer pair amplicon in the numeral correspondence on gel right side is presented at A.Swimming lane G is positive control and swimming lane N does not have genomic negative control.Molecular weight marker is in swimming lane M.The result shown represents 40 analytic samples.All 40 clone's demonstrations comprise complete genome, show that bacterial genomes is stable between conventional proliferation period in yeast.Figure 18 C provides the genomic schematic diagram of mycoplasma mycoides YCpMmycl.l; Shown the position of integrating YCp.9 independent primer pairs that use in pcr amplification their approximate position in genome show, and are numbered corresponding to the amplicon in Figure 18 B.Diagonal lines is illustrated in the amplicon of disappearance in clone 3.After transforming mycoplasma mycoides YCpMmycl.l yeast clone with the box that comprises URA3, by multiplex PCR, assess the Ura+ colone genome, and by gel electrophoresis analysis gained amplicon (data do not show).Disappearance amplicon 5 to 8 in clone 3, prompting has large disappearance in this genome.Other 4 clone's demonstrations comprise complete genome.
Figure 19 illustrates the generation of III type restriction enzyme disappearance.In order to manufacture mycoplasma mycoides III type restriction enzyme allele (typeIIIres) disappearance (iii) in yeast, by two PCR product C ORE and tandem repetitive sequence (TRS) are merged, build linear DNA fragment---knock out box (KOC) (i).This box then is transformed into and comprises the genomic yeast W303a of YCpMmyc1.1 mycoplasma mycoides bacterial strain (ii).On (-) His (-) Ura substratum, grow, to being selected via the displacement III type restriction enzyme open reading frame of 50-base pair (bp) sequence with target site (Δ typeIIIres::URA3) homology (ORF) by box.Semi-lactosi is induced and is made I-SceI endonuclease expression of enzymes, and it cuts 18-bp I-SceI site (asterisk), produces double-strand break, and this is conducive to two series connection and repeats the homologous recombination between order (TR) (the unmarked line in construct top).Restructuring between TR produces the seamless disappearance (Δ typeIIIres) of typeIIIres gene, and it is separated after the anti-selection of the 5-fluororotic acid (5-FOA) to the URA3 gene.IGR, intergenic region.Each amplicon is obtained to the size (data do not show) of wishing.
Figure 20 provides the schematic diagram that shows synthetic mycoplasma mycoides genome assembly in yeast.With three steps, from 1,078 overlapping DNA box, assemble the synthetic mycoplasma mycoides genome of 1,077,947bp.In first step, from 1 of overlapping synthetic oligonucleotide generation, 080bp box (orange arrow) is with 10 groups of restructuring, to produce 109 ~ 10kb assembly (assembly) (blue arrow).These are then with 10 groups of restructuring, to produce 11 ~ 100kb assembly (green arrow).In the terminal stage of assembling, these 11 fragments reassemble into complete genome (red circle).Except the short construct (white arrow) be stitched together of 2 vitro enzyme, by homologous recombination in body in yeast, realize assembling.Major part variation from the natural gene group is shown as yellow circle.These comprise 4 watermark region (WM1-WM4), deliberately 4kb zone (94D) and the element for shifting at yeast propagation and genome of disappearance.In addition, 20 positions (asterisk) with nucleotide polymorphisms are arranged.Genomic coordinate is relevant with the first Nucleotide of natural mycoplasma mycoides sequence.The position that has shown AscI and BssHII restriction site.Box 1 and 800-810 are optional and remove from assemble strategy.Box 2 and box 1104 are overlapping and box 799 is overlapping with box 811.
Figure 21 provides diagram by using the schematic diagram of PCR and vitro recombination correct errors.The plasmid skeleton that primer (BH pUC bckbn For1 and Rev1) is used for the recombining reaction that increases.The complement of skeleton primer (complement) (BH inset For1 and Rev1) is used in conjunction with correct errors primer (box Fix For1 and Rev1), with generation, has amplicon and the BH skeleton amplicon in homology zone each other.3 boxes that the PCR product is proofreaied and correct with generation for vitro recombination.
Detailed Description Of The Invention
A. definition
Unless otherwise defined, all technology used herein and scientific terminology have the same meaning of usually understanding as those of ordinary skills.
All patents, disclosed patent application, other publications and from the sequence of GenBank and other databases mentioned in this article, with regard to relevant technology, the integral body with them is incorporated to by reference.
Unless otherwise noted, the enforcement of the embodiment that provides will adopt the routine techniques of molecular biology etc., and it is in those skilled in the art's technical scope.These technology are fully explained in the literature.See for example Molecular Cloning:A Laboratory Mannual, (J.Sambrook etc., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (editor such as F.Ausubel, 1987 and up-to-date); Essential Molecular Biology (Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel edits, Academic Press 1991); Methods for Cloning andAnalysis ofEukaryotic Genes (editor such as Bothwell, Bartlett Publ.1990); Gene Transfer and Expression (Kriegler, Stockton Press 1990); Recombinant DNA Methodology (editor such as R.Wu, Academic Press 1989); PCR:A PracticalApproach (M.McPherson etc., IRLPress at Oxford University Press 1991); Cell Culture for Biochemists (R.Adams edits, Elsevier Science Publishers 1990); Gene Transfer Vectors for Mamalian Cells (Miller & M.Calos edits, and 1987); Mammalian Cell Biotechnology (M.Butler edits, 1991); Animal Cell Culture (editor such as Pollard, Humana Press 1990); Culture of Animal Cells, 2ndEd. (editor such as Freshney, Alan R.Liss 1987).
As used herein, " one (a) " or " one (an) " meaning is " one (one) ", " at least one " or " one or more ".
As used herein, " nucleic acid ", " nucleic acid molecule ", " nucleotide sequence ", " oligonucleotide " and " polynucleotide " are used interchangeably, and comprise Yeast Nucleic Acid (RNA) and thymus nucleic acid (DNA) the two and the nucleic acid molecule modified, nucleic acid molecule such as peptide nucleic acid(PNA) (PNA), locked nucleic acid (LNA) and other modifications, it comprises cDNA, genomic dna and mRNA and synthetic nucleic acid molecule without limitation, such as chemosynthesis or that restructuring produces those.Nucleic acid molecule can be double-stranded or strand.Wherein the nucleic acid molecule of strand can be sense strand or antisense strand.In addition, nucleic acid molecule can be annular or linear.
As used herein, " restriction endonuclease sites " refers to be limited the target nucleic acid sequence that enzyme is identified and cut.Restriction enzyme is known in the art.
As used herein, " genome " comprises that (complete) genome (for example entirely, full cell, virus and organelle gene group), and also comprise a complete genomic part, it has under at least one group of envrionment conditions, enough realize and/or maintain in cells survival (the smallest cell genome), host cell the existence (for example, minimum viral genome) of the biology that relies on host cell existence or the nucleotide sequence of organoid function (the smallest cell device genome) in host cell.Therefore, the term genome refers to full genome and is its part of minimal genome at least.Specific environment condition and the character that can specify genome to cause or maintain.In other genomic situations of organoid or viral genome or dependence host cell propagation and existence, envrionment conditions can comprise environment suitable and that the host cell of function is arranged.Therefore, the term genome comprises minimal genome and minimum replicator group and comprises over being found in the other nucleotide sequence of those nucleotide sequences in these minimal genome, but is not included in full genome all nucleotide sequences that exist.Term " genome " comprises abiogenous genome and synthetic gene group, and comprise the genetic modification genome, such as non-existent genome in laboratory and nature before, it comprises the genome of modification and comprises the hybrid gene group from the nucleic acid more than a kind and/or portion gene group.Term " genome " (for example comprises the organelle gene group, plastosome and chloroplast gene group), the genome (cellular genome) of self-replacation biology, it (for example comprises protokaryon and eukaryote, fungi, yeast, bacterium, mycoplasma), archeobacteria, vertebrates, Mammals and other biological, and viral genome and other genomes of relying on host's propagation.Genome also comprises and does not drop on how those of the biology in (Linnean) classification and synthesising biological of any known woods.Exemplary genome can be microbial genome, such as the monadic genome that comprises bacterium and yeast.
As used herein, " cellular genome " refers to comprise the genome that enough causes and/or maintain the nucleotide sequence of cells survival.These nucleotide sequences comprise that coding copies, transcribes, translation, power generation, transportation, film and cytoplasm fraction produces and those nucleotide sequences of the needed coding molecule of cell fission.Cellular genome comprises the smallest cell genome, full cellular genome and has the nucleic acid additional to the smallest cell genome but be not the genome of all nucleic acid of full cellular genome.Cellular genome is different with " organelle gene group " from " viral genome ", at least be that cellular genome comprises enough the nucleic acid for cellular replication and/or existence, and virus and organelle gene group comprise for example in host cell, maintain replication-competent virus or organoid essential but be not to maintain host cell survive or copy essential nucleic acid.
As used herein, " minimal genome " refers to by minimum nucleic acid group or the genome that basically is comprised of minimum nucleic acid group, described minimum nucleic acid group, under at least one group of envrionment conditions, enough realize and/or maintain cells survival (the smallest cell genome), in host cell, rely on host cell existence biology existence (for example, minimum viral genome) or in host cell organoid function (the smallest cell device genome).Should be understood that even full organelle gene group there is no need coding and makes organoid preserve needed all proteins, but some protein are by the genes encoding in the nucleus of the cell comprising organoid.Therefore, the smallest cell device genome only needs to be included in cellular environment those genes essential for the organoid function.Similarly, should be understood that virus relies on host cell existence, therefore minimum viral genome only needs to support the existence of virus in host cell." minimum replicator group " is except the minimum nucleotide sequence of enough surviving, and also comprises the minimal genome of enough nucleotide sequences for cell or biological self-replacation.
As used herein, synthetic nucleic acid sequence, comprise the synthetic gene group, and its all or part builds from the copy of genetic module or these assemblies, and described genetic module is chemosynthesis in vitro.Copy can produce by any many methods known in the art, comprises in body and body outer clone and amplification method.Complete synthetic nucleotide sequence or genome is such: wherein whole nucleic acid or genome are by chemosynthesis or generation or assembling from the nucleic acid copy of these external chemosynthesis in vitro.By comparison, " semi-synthetic " genome refers to nucleotide sequence or the genome of partial synthesis, and it is that the some of them genetic module is abiogenous synthetic gene group, comprises the nucleic acid of cloning from abiogenous nucleic acid.
As used herein, external source or heterologous gene group or nucleotide sequence are to have genome or the nucleotide sequence still come from the different types of donor biology of host cell in host cell.The donor biology can be different genus, order, boundary or other genetic typing, or can be simply not of the same race in same genus.
As used herein, " target nucleic acid sequence " refer to, is the nucleotide sequence of the target of for example being modified by modifying method described herein and known in the art.One or more modifications of target nucleic acid sequence comprise introduces target nucleic acid sequence by one or more sudden changes, one or more disappearance, one or more replacement and/or one or more insertion.Target region is the specific region of target nucleic acid sequence, such as single locus, polygene seat or its part as modifying object.In a kind of example, target region comprises the zone of target nucleic acid sequence, and it is such as for example being replaced by homologous recombination with another nucleotide sequence.Target nucleic acid sequence is compared with the primary target zone after modifying, and in the nucleotide sequence of modifying, there is no need to modify whole target regions.For example, the modification of target region can be included in that target position in target region/residue place singly inserts, disappearance or replace, and maybe can be included in the modification of many position/residues of one or more target parts of target region.
B. for cloning and operate the method for genome and large nucleic acids
This paper provides nucleic acid, method and system, it is for introducing the heterologous host cell by donor gene group and other donor nuclei acid sequences, for at host cell, modifying donor gene group and nucleotide sequence, reclaim donor gene group and nucleotide sequence and the donor gene group and the nucleotide sequence that reclaim are introduced to recipient cell from host cell.Be included within the scope of the nucleotide sequence that provides, method and system be uncompatibility between nucleotide sequence, cell and the genetic system that makes donor, host and acceptor for example and/or the minimized aspect of toxicity.The illustrative embodiments of institute's supplying method is illustrated in Fig. 1, wherein the method is used for by bacterium donor gene group is connected with the yeast host carrier and makes bacterium donor gene group be transformed into yeast host cell, modify the donor gene group and shift the donor gene group of modifying in yeast host cell and enter the bacterial receptor cell, thereby, produce the engineering bacterium.As indicated in Fig. 1, the modifying factor group existed in the engineering bacterium can be separated and in the round subsequently of method with iterative manner as the donor gene group.As described herein, consider many modification of this embodiment, and drop in the application's scope.Another illustrative methods of institute's supplying method is illustrated in Figure 16, wherein the method is for inserting bacterial genomes by host's yeast vector, the genome that separates the yeast vector with integration, with bacterial genomes/yeast vector transformed yeast host cell, modify bacterial genomes, separate the genome of modifying, optionally methylate genome and transfer donator genome enter recipient cell.
Embodiment 5 has described the successful combination of institute's supplying method, by the donor bacterium genome is transformed into to yeast host cell, modify the donor gene group in yeast cell, and the donor gene group that then shifts the gained modification enters recipient cell, so the genetic expression from the donor gene group of modifying is induced, and has produced new bacterium living beings.By genomic sequence verification in negative control and recipient cell this result.This research shows successfully to have generated the mycoplasma mycoides LC genome that comprises the seamless disappearance of III type restriction enzyme allele, its in mycoplasma, use state-of-the art method, use its available natural genetic system to generate.Transfer has produced non-existent mycoplasma mycoides LC bacterial strain in laboratory and nature before.Therefore, the method provided is used in the genomic extracellular of being transformed of encoding and successfully carries out genetic modification.
Method and kit for and resulting cell for operating genome and large nucleic acids are provided, for example to transform and to change, can have produced the biology of useful compound such as vaccine, medicine, biogenic protein or chemical and biofuel.The weak genetic system of biology method has to(for) operation is such as genome and the karyomit(e) of unmanageable biology especially need, and to produce gene, genome and the biology of modifying, it produces new gene product such as to power generation and useful those of medical science.
Before the application, for the methods availalbe that operates genome and other large nucleic acids, be limited.Have the character/characteristic of expectation/phenotype such as the ability that produces useful compound and the ability that can work at extreme environment comprise that unicellular organism is such as procaryotic many biologies, have very weak or there is no a genetic system (allowing the system of the nucleic acid of modified biological in laboratory).Therefore, the disclosure provides these genomes and nucleic acid has been transferred to other cells (host cell) with genetic system of more expecting system modifying factor group and the needed Method and kit for of nucleic acid that uses expectation in these other cells.
In order to produce new gene product the nucleic acid from modifying, the present invention also provides as this method: they are shifted and enter the environment of expressible gene product therein from host cell, such as suitable recipient cell.For example, although it may be enough expressing in host cell, but may expect that the donor gene group moves up into recipient cell from the host cell transfer, described recipient cell has the cellular environment of the initial donorcells that is derived to natural or synthetic donor gene group or biological cellular environment more similar (replicate).Generally speaking, this paper provides and can be used for operation and modifying gene group and other nucleic acid especially transfer, clone, modification and transfer method, nucleic acid and the system of the raising of large nucleic acids.
Similarly, before the application, by available method, shift and comprise that it is limited that genomic large nucleic acids enters host cell.Although the ordinary method for cloning nucleic acid is (bacterium and yeast with good genetic system are used and acted on from the host of many biology cloning nucleic acid fragments) known, the restriction relevant to these methods can make them not be expected to be useful in operation and modifying gene group and large nucleic acids.For example, the nucleic acid size of using ordinary method can be cloned into host cell is limited.The nucleic acid of cloning by ordinary method generally comprises only minority gene.
Uncompatibility and toxicity problem also can limit available cloning process.For example, donor nucleic acid to host cell can be poisonous (for example, if poisonous protein express from donor nucleic acid) and in event such as the genetic system that uses the host carries out during host cell copies and/or modifies becoming unstable.This class event can be limited in the ability of operation nucleic acid in host cell.In addition, the modification of not expecting to the donor gene group that can occur during modification does not have the fact of negative impact usually to the existence of host cell, can make donor gene group and other nucleic acid unstable in host cell.
The uncompatibility problem also can be damaged the donor gene group and shift the environment get back to more natural gene product expression from host cell, such as the efficiency of the recipient cell that enters or the kind that be closely related identical with donor.In provided embodiment, modifying method has overcome these problems, for different host cell in heredity, such as the yeast host cell that enters the recipient cell more different from the kind of initial donor gene group for transfer is successfully bred and modifies the donor nucleic acid that comprises genome.
Reclaim the donor gene group and further donor gene group introducing recipient cell can be caused to other challenge from host cell, wherein donor, host and recipient cell are (such as the biological branches from different) be not closely related.For example, due to uncompatibility and toxicity problem, the donor gene group that will breed in eucaryon host is introduced former nuclear receptor and may be limited.If the donor gene group is bred in host cell, the restriction modification system that exists (perhaps also existing in donorcells) still not exist in host cell in recipient cell can cause uncompatibility after transfer.Although some host cells, such as yeast does not comprise restriction modification system, but they can express the dnmt rna that can be modified at the donor nucleic acid of breeding in host cell, thus the activation of inhibition donor nucleic acid (for example, genome) after transfer enters recipient cell.In host cell propagation and modify after, the structure of the donor gene group of separation and conformation also can be different from conformation and the structure of the homologous genes group of breeding in cell more being closely related with the donor biology.This species diversity can adversely affect transfer.
Method described herein has overcome these restrictions, for successfully cloning the donor gene group, in host cell, modifies and/or propagation donor gene group, reclaims the donor gene group and the donor gene group is introduced to recipient cell.On the one hand, the donor gene group reclaimed from host cell is introduced into the upper different recipient cell (such as from eucaryon host to former nuclear receptor) of heredity.
The donor nuclei acid sequence, for example, the donor gene group is selected and synthesizes, assemble and/or separate (for example,, from donorcells) and be cloned into host cell.The method comprises with one or more fragment from donorcells acquisition donor gene group or synthetic donor gene group, with donor gene group and host's carrier are introduced to the heterologous host cell, wherein optionally the donor gene group is connected with host's carrier, then introduce host cell, thereby generate the host cell that comprises the donor gene group, described donor gene group comprises host's carrier, and further wherein the donor gene group is basically complete cell, virus or organelle gene group, it is at least minimal genome, and length is greater than about 300kb.
The first embodiment typically relates to introduces by host's carrier the cell that comprises the donor gene group, the donor gene group is connected with host's carrier, donor gene group transformed host cell that recovery comprises host's carrier, so that during the donor gene group that comprises host's carrier remains on host cell between the host cell replicative phase.
In the second embodiment, in host cell, host's carrier and linearizing donor gene group are by cotransformation, and wherein in host cell, host's carrier is connected by homologous recombination with the donor gene group.
In the third embodiment, overlapping DNA fragment in host cell (natural or synthetic) and host's carrier are by cotransformation, and wherein in host cell, host's carrier is connected by homologous recombination with DNA fragmentation.
The donor gene group of using in the method can be full cell, virus or organelle gene group basically.
Donor gene group and host's carrier can simultaneously or be introduced host cell with arbitrary order in succession.In one embodiment, host's carrier is connected with the donor gene group, then by transforming host's carrier, enters the donorcells introducing host cell that comprises the donor gene group.
The host cell used in the present embodiment can be eukaryotic cell or prokaryotic cell prokaryocyte.Host cell includes but not limited to, bacterial cell, fungal cell, insect cell, vegetable cell or alga cells.Host cell also comprises yeast cell.
Host's carrier is the carrier that can be used for homologous recombination.The host's carrier used in the present embodiment can be the kinetochore plasmid.In one embodiment, host's carrier is that yeast centromeric plasmid and host cell are yeast cell.
Consider that in the present embodiment the donor gene group of using can be, for example bacterial genomes, fungal gene group, Yeast genome, archeobacteria genome, cyanobacteria genome, algae genome, phage genome, Mitochondrial Genome Overview, chloroplast gene group, viral genome, organelle gene group or synthetic gene group.
On the other hand, method also is included in host cell and modifies the donor gene group.
On the other hand, method also comprises the donor gene group that recovery comprises host's carrier from host cell.
On the other hand, method also comprises the donor gene group introducing recipient cell that will reclaim.
On the other hand, method also comprises degraded or the native gene group of removing recipient cell.
Optionally, the donor gene group of recovery can be methylated before entering recipient cell.
Optionally, the restriction enzyme function of recipient cell be non-existent, be removed or inactivation.
Consider that in the present embodiment the recipient cell used can be, for example bacterial cell, yeast cell, fungal cell, insect cell, vegetable cell or alga cells.
On the other hand, method also comprises introduces host cell by the second donor gene group, and wherein the second donor gene group is different from the first donor gene group, therefore produces the host cell that comprises two different donor gene groups.Introducing the second donor gene group can comprise and make the host cell that comprises the first donor gene group and the second host cell mating that comprises the second donor gene group.But the donor gene group of recovery is introduced on the recipient cell phenotype recipient cell is changed into and the corresponding phenotype of the donor gene that is incorporated to any modification.
This paper provides the cell of separation, the synthetic or restructuring produced by any method described herein.
This paper provides the method for the manufacture of cell, described cell display is by the phenotype of donor gene group coding, described method comprises: (a) donor gene group and the host's carrier that is adapted at clone's donor gene group in host cell are introduced to host cell, obtain like this product that comprises the donor gene group, described donor gene group comprises host's carrier; (b) be recovered in the product that comprises the donor gene group obtained in step (a) from host cell, described donor gene group comprises host's carrier; (c), the product of (b) is introduced to recipient cell under certain condition, so that recipient cell is showed the phenotype by this product coding; (d) reclaim the cell obtained from step (c); Wherein the donor gene group is basically complete cell, virus or organelle gene group, it is at least minimal genome, and the minority component that length is greater than about 300kb and comprises nucleic acid substances, described minority component shows that for recipient cell the phenotype by the donor gene group coding is essential.On the one hand, method also is included in the donor gene group of modifying (a) in host cell.On the other hand, method also comprises degraded or the native gene group of removing recipient cell.Another another aspect, method also is included in the donor gene group of modifying (a) in host cell the native gene group of degrading or removing recipient cell.
This paper provides the cell of showing the expectation phenotype, described expectation phenotype by the donor gene group coding and otherwise can be by this cell display, wherein cell is produced by method described herein.
This paper also provides the cell that comprises the donor gene group and show the expectation phenotype, described expectation phenotype by the donor gene group coding and otherwise can be by cell display, wherein the donor gene group comprises the foreign gene group nucleic acid substances that is greater than 300kb and for the essential genome minority component of cell display expectation phenotype.
Modifying method and instrument comprise make during modifying the instable risk minimization of donor nuclei acid sequence in host cell aspect.In the third embodiment, the donor nuclei acid sequence moves up into recipient cell from the host cell transfer, and described recipient cell can be the kind different from donorcells and host cell and/or different biological branches, or the kind identical with donorcells.Transfer method comprises the aspect that makes the risk minimization of uncompatibility and toxicity between donor gene group, host cell and recipient cell.
Can separately be shifted, modification and transfer method, also capable of being combinedly in succession carry out.Therefore, in one embodiment, three steps can be combined in a method, be transferred and enter host cell by the method donor gene group, modify in host cell and shift and enter recipient cell, to produce new cell, thereby generate non-existent genome or cell in laboratory and nature before.But the recipient cell further growth becomes in non-existent non-human organism before or is transferred to wherein.Therefore, the method provided, nucleic acid and system can be used for producing new biology.New-create biology and its nucleotide sequence also are provided.
The method and composition provided is particularly useful for the genome operated and transform from unmanageable biology in heredity.In a kind of example, method, nucleotide sequence and system are for cloning as ring-type kinetochore plasmid the full bacterial genomes from mycoplasma genitalium, mycoplasma pneumoniae (Mycoplasma pneumoniae) and mycoplasma mycoides LC at yeast, modify the donor gene group to use the yeast genetic system with modification in yeast, so that uncompatibility minimizes, further the bacterial genomes of modification is shifted and enters different types of recipient cell, thereby generate non-existent genome and biology in laboratory and nature before.
The method provided, nucleotide sequence, system and the biological biology that can be used for transforming synthesising biological fuel.For example, although bacterium can be by genetic modification such as intestinal bacteria (Escherichia coli), there is the potentiality that produce industrial useful compound or many prokaryotic organism of working at extreme environment and have very weak or there is no a genetic system.Proto green algae (Prochlorococcus marinus) is one of photosynthetic organism the abundantest on the earth.Although desired operation is also transformed this and other such biologies to produce biofuel, owing to lacking their methods availalbe of genetic modification, operation and the transformation ability of biology like this are limited.The method provided can be used for carrying out these operations.For example, in one embodiment, the nucleotide sequence of the pathways metabolism component of encoding new can be introduced these biological genomes and modify in host cell by transfer.The genome of this transformation again is transferable enters suitable recipient cell, to produce new cell, for example, can make sunlight become the new cell of biofuel with carbon dioxide conversion.This paper also provides the Cell and organism of this class transformation.
The method provided also can be used for transforming archeobacteria, clone new organoid enters eukaryote and adds karyomit(e) in Cell and organism.For example, eucaryon plastosome and chloroplast(id) are the residues of captive endosymbiosis bacterium in their hosts.The method provided is used in the host, for example, in the yeast that uses plasmid, use homologous recombination to transform these organelle gene groups, thereby such as create power generation efficiency and/or metabolic new plastosome and the chloroplast gene group with raising in yeast or algae.
In another embodiment, the method provided can be used for operation virus, such as having for operating and large genomic those viruses of Yan Taida in simple plasmid, has virus and the phage for the treatment of application with generation.On the one hand, viral genome is cloned and operates to improve their immunogenicity and other treatment advantage.
In another embodiment, the method provided can be used for operating fungi and for example produces useful fungi in wine, bread, beer and medicine to be created in.On the one hand, clone and operation fungal gene group are to improve them to the resistance of temperature, pathogenic organisms and other advantages.In another embodiment, the method provided can be used for operating yeast to produce for alcohol fuel, nutritional additive, probiotic agent, the fermentation of producing for beverage (alcohol type and non-alcohol type) or to be used in the useful yeast of fermentation cured.
Although this paper provides some embodiment, Method and process of the present invention is the general utility tool that can be used for producing any expectation phenotype or product of interest.
Method and process of the present invention is easy to automatization and adapts to high-yield method, for example, the computer regulating by not needing the people to intervene and/or automatic mode can many nucleic acid molecule be connected simultaneously and conversion enter host or recipient cell.
Therefore, the present invention relates to method and the product thereof of system, its permission effectively and is in large quantities assembled, clones, modifies and transform the nucleic acid molecule that comprises genome in the mode of high yield, and easily is applicable to automatization enforcement.In optional embodiment, the nucleic acid assembly reaction can carry out on solid surface---for example, with contrary in reaction tubes, on the chip that uses microfluid.
C. select and separate donor gene group and nucleic acid
In the first step of institute's supplying method, select donor gene group or other nucleotide sequences for shifting, modifying and/or shift.The nucleotide sequence that shifts, modifies, shifts and generate by method described herein can be the nucleotide sequence of any natural or synthesising biological.Therefore, the donor gene group is by separating or come from by chemosynthesis cell or its any subunit that comprises nucleic acid of any expectation.For example, nucleotide sequence comprises from known organism or neoplastic genome (such as full genome, be at least a complete genomic part, cellular genome, organelle gene group and the viral genome of minimal genome and/or at least minimum replicator group), karyomit(e) and other large nucleic acids sequences.Nucleotide sequence, comprise genome, it can be any source in biology, comprise the organelle gene group, such as the genome of plastosome and chloroplast gene group, karyomit(e), plant and animal or a chromosomal part, alga-derived and support any genomic material of cells survival, it comprises bacterium and other prokaryotic organism and Eukaryotic full cellular genome and the smallest cell genome.
Obvious from the commentary of following embodiment and discussion provided herein, the suitability of institute's described method is not limited to build the synthetic gene group existed in natural imitation.Method can be used for, and for example different biological portion gene groups is connected in the same DNA molecule, to be created on non-existent new genome and biology in nature or laboratory.The donor gene group be cloned, breed with other nucleic acid and/or from cell such as cell or tissue (comprising the genetic modification biology) separates, or chemosynthesis in vitro.The following describes for separating of with prepare nucleic acid and genomic method.
I. biological, the genome of donor and other nucleic acid
The genome that uses and generate in provided method and other nucleotide sequences (for example, donor nucleic acid) comprise and come from fungi, yeast, bacterium, other prokaryotic organism and algae but be not limited to those of these biologies.They can be any biologies, natural or synthetic, for example biology and the virus on protobiont (Protista) boundary, archeobacteria (Archaebacteria) boundary, eubacterium (Eubacteria) boundary, fungi (Fungi) boundary, plant (Plantae) boundary and animal (Animalia) boundary---comprise phage.
The Exemplary core acid sequence is (for example to come from bacterium, archeobacteria, cyanobacteria, proto green algae, cytoalgae PCC6803 etc.), algae, virus (for example, the hemophilus influenzae genome), those of fungi (for example, yeast saccharomyces cerevisiae, saccharomyces bayanus (Saccharomyces bayanus), Bradley yeast (Saccharomyces boulardii), Neurospora crassa (Neurospora crassa) etc.) and phage.Exemplary mycoplasma bacterial strain comprises that mycoplasma genitalium (for example, mycoplasma genitalium bacterial strain MS5, the mycoplasma genitalium G37 (No. GenBank: L43967)), mycoplasma mycoides (Mycoplasma mycoides) (for example, the large colony of mycoplasma mycoides subspecies mycoides (LC) bacterial strain GM 12 (embodiment 1), mycoplasma capri (Mycoplasma capricolum) subspecies capricolum (the bacterial strain California Kid that in embodiment 1, describe TM) (ATCC 27343), mycoplasma mycoides subspecies mycoides (bacterial strain GM12) (Damassa etc., 1983), mycoplasma capri subspecies capricolum (mycoplasma capri) for example, such as wild-type mycoplasma capri and mycoplasma capri mutant (mycoplasma capri-Δ RE) and mycoplasma pneumoniae (, mycoplasma pneumoniae bacterial strain M129-B170 (ATCC 29343); Mycoplasma pneumoniae M129, GenBank accession number U00089.2 (GI:26117688)), Mycoplasma gallisepticum (ATCC15302), mycoplasma pneumoniae Eaton (ATCC15531) and derivative thereof.
Exemplary genome and nucleic acid comprise the complete of many biologies and portion gene group, and its genome sequence is disclose available and can be used for method of the present disclosure, is such as but not limited to super Pyrococcus furiosus (Aeropyrum pernix); Agrobacterium tumefaciens (Agrobacterium tumefaciens); Anabena (Anabaena); Gambia malarial mosquito (Anopheles gambiae); Apis mellifera (Apis mellifera); Hyperthermophile (Aquifex aeolicus); Arabic mustard (Arabidopsis thaliana); Super hyperthermophilic archaeon strain (Archaeo globus fulgidus); The yellow bacterium (Ashbya gossypii) of core; Bacillus anthracis (Bacillus anthracis); Bacillus cercus (Bacillus cereus); Alkaliphilic bacillus (Bacillus halodurans); Bacillus licheniformis (Bacillus licheniformis); Hay bacillus (Bacillus subtilis); Bacteroides fragilis (Bacteroides fragilis); Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron); Sieve Heng Shi Cali martensite bacterium (Bartonella henselae); Bartonella quintana (Bartonella Quintana); Bdellovibrio (Bdellovibrio bacteriovorus); Bifidobacterium longum (Bifidobacterium longum); Blochmannia floridanus; The special bacterium (Bordetella bronchiseptica) of bronchitis Boulder; Parapertussis bacillus (Bordetella parapertussis); Bordetella pertussis (Bordetella pertussis); Borrelia burgdoyferi (Borrelia burgdorferi); Rihizobium japonicum (Bradyrhizobium japonicum); Brucella melitensis (Brucella melitensis); Brucella suis (brucella suis); Aphid endosymbiosis bacterium (Buchnera aphidicola); Pseudomonas mallei (Burkholderiamallei); Pseudomonas Pseudomallei (Burkholderia pseudomallei); Nematode kind (Caenorhabditisbriggsae); Caenorhabditis elegans (Caenorhabditis elegans); Campylobacter jejuni (Campylobacterjejuni); Candida glabrata (Candida glabrata); Domesticated dog (Canis familiaris); Crescent handle bacillus (Caulobacter crescentus); Mouse type chlamydia trachomatis (Chlamydis muridarum); Chlamydia trachomatis (Chlamydis trachomatis); Cavy preferendum Chlamydia (Chlamydophila caviae); Chlamydophila pneumoniae (Chlamydophila pneumoniae); Chloracea (Chlorobium tepidum); Chromobacterium violaceum (Chromobacterium violaceum); Ciona (Ciona intestinalis); Acetone-butanol Fusobacterium (Clostridium acetobutylicum); C.perfringens (Clostridium perfringens); Clostridium tetani (Clostridium tetani); Corynebacterium diphtheriae (Corynebacterium diphtheriae); Effective excellent bacillus (Corynebacterium efficiens); Bai Shi cock steadite (Coxiella burnetii); People Cryptosporidium (Cryptosporidium hominis); Cryptosporidum parvum (Cryptosporidium parvum); Cryptosporidium parvum (Cyanidioschyzon merolae); Debaryomyces hansenji (Debaryomyces hansenii); Radioresistant cocci (Deinococcus radiodurans); Desulfotalea psychrophila; Desulfovibrio (Desulfovibriovulgaris); Drosophila melanogaster (Drosophila melanogaster); Encephalitozoon (Encephalitozoon cuniculi); Enterococcus faecalis (Enterococcus faecalis); Carrot soft rot Erwinia (Erwinia capotovora); Escherichia coli (Escherichia coli); Fusobacterium nucleatum (Fusobaceterium nucleatum); Hongyuan chicken (Gallus gallus); Geobacter metallireducens (Geobacter sulfurreducens); Without thylakoid blue-green algae (Gloeobacter violaceus); Lan Yinzao (Guillardia theta); Haemophilus ducreyi (Haemophilus ducreyi); Haemophilus influenzae; Extremely halophilic archaea (Halobacterium); Helicobacter hepaticus (Helicobacter hepaticus); Helicobacter pylori (Helicobacter pylori); Homo sapiens (Homo sapiens); The male saccharomycete (Kluyveromyces waltii) of Crewe; Yue Shi lactobacillus (Lactobacillus johnsonii); Lactobacillus plantarum (Lactobacillus plantarum); Legionella pneumophilia (Legionellapneumophila); Sugarcane ratoon stunting disease pathogen (Leifsonia xyli); Lactococcus lactis (Lactococcus lactis); Leptospira (Leptospira interrogans); Harmless Li Site bacterium (Listeriainnocua); Listerisa monocytogenes in mjme (Listeria monocytogenes); Pyricularia oryzae (Magnaporthe grisea); Produce butanedioic acid Man bacillus (Mannheimia succiniciproducens); Mesoplasma florum; Living rhizobium in crowtoe (Mesorhizobium loti); Methane bacteria (Methanobacterium thermoautotrophicum); Have a liking for cold methanogen (Methanococcoidesburtonii); Methanococcus jannaschii (Methanococcus jannaschii); Sea natural pond methanosarcina (Methanococcus maripaludis); Cold-resistant methane backeria (Methanogenium frigidum); Methane Thermophilic Bacteria (Methanopyrus kandleri); Acetic acid sarcina methanica (Methanosarcina acetivorans); Ma Shi sarcina methanica (Methanosarcina mazei); Have a liking for methane backeria (Methylococcus capsulatus); House mouse (Mus musculus); Mycobacterium bovis Mycobacterium bovis); Mycobacterium leprae (Mycobacteriumleprae); Mycobacterium paratuberculosis (Mycobacterium paratuberculosis); Tubercle bacillus (Mycobacterium tuberculosis); MG (Mycoplasma gallisepticum); Mycoplasma genitalium (Mycoplasma genitalium); Mycoplasma mycoides (Mycoplasma mycoides); Myoplasna penetrans (Mycoplasma penetrans); Mycoplasma pneumoniae (Mycoplasma pneumoniae); Mycoplasma pulmonis (Mycoplasma pulmonis); Mycoplasma mobile (Mycoplasma mobile); Receive ancient bacterium (Nanoarchaeumequitans); Neisseria meningitidis (Neisseria meningitidis); Neurospora crassa (Neurospora crassa); Europe Nitrosomonas (Nitrosomonas europaea); Pi Nuoka bacterium (Nocardia farcinica); Bridge ocean, Yi Ping room bacillus (Oceanobacillus iheyensis); Onion yellow phytoplasma (Onions yellowsphytoplasma); Paddy rice (Oryza sativa); Chimpanzee (Pan troglodytes); Pasteurella multocida (Pasteurella multocida); Phanerochaete chrysosporium (Phanerochaete chrysosporium); Luminous bacillus (Photorhabdus luminescens); Xeothermic acidophil (Picrophilus torridus); Plasmodium (Plasmodium falciparum); Mouse plasmodium (Plasmodium yoelii yoelii); Comospore poplar (Populus trichocarpa); Porphyromonas gingivalis (Porphyromonas gingivalis); Prochlorococcus; Propionibacterium (Propionibacterium acnes); Protochlamydia amoebophila; Pseudomonas aeruginosa (Pseudomonas aeruginosa); Pseudomonas putida (Pseudomonas putida); Pseudomonas syringae (Pseudomonas syringae); Aerophil (Pyrobaculum aerophilum); Seabed fireball bacterium (Pyrococcus abyssi); High Pyrococcus furiosus (Pyrococcus furiosus); Extreme hyperthermophilic archaeon strain (Pyrococcus horikoshii); Fumaric acid fire leaf bacterium (Pyrolobus fumarii); Ralstonia solanacearum (Ralstonia solanacearum); Rattus norvegicus (Rattus norvegicus); Rhodopirellula baltica; Rhodopseudomonas palustris (Rhodopseudomonas palustris); Dermacetor conori (Rickettsia conorii); Rickettsia exanthematotyphi (Rickettsia typhi); Rickettsia prowazeki (Rickettsia prowazekii); Dermacetor sibericus (Rickettsia sibirica); Saccharomyces cerevisiae; Saccharomyces bayanus (Saccharomyces bayanus); Bradley yeast (Saccharomyces boulardii); The sugar red moulds of many spores (Saccharopolyspora erythraea); Intestines salmonella (Salmonella enterica); Salmonella typhimurtum (Salmonella typhimurium); Grain brewer yeast (Schizosaccharomyces pombe); A kind of apple difficult to understand reaches Shewanella (Shewanella oneidensis); Shigella dysenteriae (Shigella flexneria); Rhizobium melioti (Sinorhizobium meliloti); Staphylococcus aureus (Staphylococcus aureus); MRSE (Staphylococcus epidermidis); Streptococcusagalactiae (Streptococcus agalactiae); Streptococcus mutans (Streptococcus mutans); Streptococcus pneumonia (Streptococcus pneumoniae); Streptococcus pyogenes (Streptococcus pyogenes); Streptococcus thermophilus (Streptococcus thermophilus); Avid kyowamycin (Streptomyces avermitilis); Streptomyces coelicolor (Streptomyces coelicolor); Have a liking for the ancient bacterium (Sulfolobus solfataricus) of superhigh temperature; Super hyperthermophilic archaeon strain (Sulfolobus tokodaii); Synechococcus (Synechococcus); Cyanobacteria (Synechocystis); Fugu rubripes (Takifugu rubripes); Black blue spot filefish (Tetraodon nigroviridis); Thalassiosira pseudonana (Thalassiosira pseudonana); Tengchong thermophilic bacteria (Thermoanaerobacter tengcongensis); Thermoplasma acidophilum (Thermoplasma acidophilum); Hot volcanic substance (Thermoplasma volcanium); Thermotoga maritima (Thermotagoa maritima); Thermus thermophilus (Thermus thermophilus); Tartar treponema (Treponema denticola); Spirochaeta pallida (Treponema pallidum); You support barrier body (Tropheryma whipplei) positive Hewlett-Packard; Ureaplasma urealyticum (Ureaplasma urealyticum); Comma bacillus (Vibrio cholerae); Vibrio parahaemolytious (Vibrio parahaemolyticus); Vibrio vulnificus (Vibrio vuinificus); Wigglesworthia glossinidia; Fertile Bach Salmonella (Wolbachia pipientis); Produce the fertile honest and clean bacterium (Wolinella succinogenes) of butanedioic acid; Citrus ulcer bacteria (Xanthomonas axonopodis); Black rot (Xanthomonas campestris); Xyllela fastidiosa (Xylella fastidiosa); Fat Ye Shi yeast (Yarrowia lipolytica); Yersinia pseudotuberculosis (Yersinia pseudotuberculosis); And yersinia pestis (Yersinia pestis) nucleic acid.
Term " algae " comprises cyanobacteria (Cyanophyceae (Cyanophyceae)), green alga (Chlorophyceae (Chlorophyceae)), yellowish green algae (Xanthophyceae (Xanthophyceae)), chrysophyceae (Chrysophyceae (Chrysophyceae)), brown alga (Phaeophyceae (Phaeophyceae)), red algae (Rhodophyceae (Rhodophyceae)), diatom (Diatomacae (Bacillariophyceae)) and " ultraplankton " (blue or green Chlorophyceae (Prasinophyceae) and true whip algae guiding principle (Eustigmatophyceae)).The term algae also comprises taxonomy Dinophyceae (Dinophyceae), Cryptophyceae (Cryptophyceae), Euglenophyceae (Euglenophyceae), grey algae guiding principle (Glaucophyceae) and general woods algae guiding principle (Prymnesiophyceae).Microalgae is the unicellular or group algae that only just can see under microscopical help.Microalgae comprises Eukaryotic Algae and protokaryon algae (for example, cyanobacteria).Photosynthetic bacteria comprises cyanobacteria, green sulphur bacteria, purple sulfur bacterium, purple nonsulfur bacteria and green without thiobacterium.
Exemplary genome and nucleic acid comprise the complete of many algae bios and portion gene group, and its genome sequence is disclose available and can be used for method of the present disclosure, is such as but not limited to Achnanthes (Achnanthes), cocoon shape Trentepohlia (Amphiprora), double eyebrow algae spp (Amphora), Ankistrodesmus (Ankistrodesmus), star born of the same parents Trentepohlia (Asteromonais), Boekelovia, Borodinella, grape Trentepohlia (Botryococcus), lactococcus (Bracteococcus), Chaetoceros belongs to (Chaetoceros), Tetrablepharis (Carteria), Chlamydomonas (Chlamydomonas), Chlorococcum (Chlorococcum), green shuttle Trentepohlia (Chlorogonium), Chlorella (Chlorella), Chroomonas (Chroomonas), gold goal Trentepohlia (Chrysosphaera), ball calcium plate Trentepohlia (Cricosphaera), Crypthecodinium cohnii belongs to (Crypthecodinium), hidden Trentepohlia (Cryptomonas), Cyclotella (Cyclotella), Halophila (Dunaliella), oval Trentepohlia (Ellipsoidon), ballstone Trentepohlia (Emiliania), only ball Trentepohlia (Eremosphaera), Ernodesmius, Euglena (Euglena), drape over one's shoulders thorn Trentepohlia (Franceia), drape over one's shoulders thorn Trentepohlia (Fragilaria), Liz Trentepohlia (Gloeothamnion), haematococcus Haematococcus), Halocafeteria, hymenomonas (Hymenomonas), Isochrysis galbana belongs to (Isochrysis), Lepocinclis (Lepocincls), Micractinium pusillum belongs to (Micractinium), single needle Trentepohlia (Monoraphidium), microballoon algae (Nannochloris), micro-algae (Nannochloropsis), boat-shaped algae (Navicula), fresh green Trentepohlia (Neochloris), kidney Dictyocha (Nephrochloris), Nephroselmis (Nephroselmis), prismatic Trentepohlia (Nitzschia), Ochromonas (Ochromonas), Oedogonium (Oedogonium), egg capsule Trentepohlia (Oocystis), Ostreococcus, bar husband's Trentepohlia (Pavlova), Parachlorella, Pascheria, brown algae belongs to (Phaeodactylum), Phagus (Phagus), flat algae belongs to (Platymonas), cocolith (Pleurochrysis), rib ball Trentepohlia (Pleurococcus), Prototheca (Prototheca), Pseudochlorella, tower born of the same parents algae (Pyramimonas), mulberries Trentepohlia (Pyrobotrys), Scenedesmus (Scenedesmus), schizochytrium limacinum belongs to (Schizochytrium), Skeletonema (Skeletonema), Spyrogyra, split Ulothrix (Stichococcus), merge micro-algae (Tetraselmis), thraustochytriale (Thraustochytrium), Thalassiosira (Thalassiosira), Viridiella or Volvox (Volvox) kind.In some embodiments, can use photosynthetic bacteria, for example comprise, green sulphur bacteria, purple sulfur bacterium, green without thiobacterium, purple nonsulfur bacteria or cyanobacteria.Spendable cyanobacteria kind includes but not limited to sheet algae (Agmenellum, Anabaena (Anabaena), necklace Trentepohlia (Anabaenopsis), Anacystis nidulans belongs to (Anacystis), Aphanizomenon (Aphanizomenon), Arthrospira (Arthrospira), Asterocapsa (Asterocapsa), Borzia (Borzia), Calothrix (Calothrix), pipe spore Trentepohlia (Chamaesiphon), sticky belong to (Chlorogloeopsis) of green rod, mimic colouration ball Trentepohlia (Chroococcidiopsis), chromosphere Trentepohlia (Chroococcus), scared pin Trentepohlia (Crinalium), cyanobacteria (Cyanobacterium), blue Pseudomonas (Cyanobium), blue cyst Pseudomonas (Cyanocystis), blue spirillum (Cyanospira), blue bar Trentepohlia (Cyanothece), intend post spore Trentepohlia (Cylindrospermopsis), cylinder spore Trentepohlia (Cylindrospermum), plan refers to ball Trentepohlia (Dactylococcopsis), cuticle fruit cyanobacteria belongs to (Dermocarpella), Fei Shi Trentepohlia (Fischerella), Fremyella, lucky Teller Bordetella (Geitleria), Geitlerinema, Bacillus adhaerens belongs to (Gloeobacter), Ku Shi slimeball algae (Gloeocapsa), sticky bar Trentepohlia (Gloeothece), salt Spirullina (Halospirulina), Iyengariella, thin sheath Ulothrix (Leptolyngbya), blue Ulothrix (Limnothrix), sheath Ulothrix (Lyngbya), Microccoleus (Microcoleus), Chlorella (Microcystis), Myxosarcina (Myxosarcina), joint ball Trentepohlia (Nodularia), Nostoc (Nostoc), intend pearl Trentepohlia (Nostochopsis), Oscillatoria (Oscillatoria), Phormidium (Phormidium), floating Ulothrix (Planktothrix), wide ball Trentepohlia (Pleurocapsa), proto green algae belongs to (Prochlorococcus), former green alga belongs to (Prochloron), green Trentepohlia (Prochlorothrix), Pseudanabaena sp belongs to (Pseudanabaena), glue palpus Trentepohlia (Rivularia), split palpus Trentepohlia (Schizothrix), Scytonema (Scytonema), Spirullina (Spirulina), Stanieria, this Ta Ershi cyanobacteria belongs to (Starria), multiple row Trentepohlia (Stigonema), bundle Trentepohlia (Symploca), collection ball Trentepohlia (Synechococcus), synechocystis (Synechocystis), Tolypothrix (Tolypothrix), Trichodesmium (Trichodesmium), Tychonema or different ball Trentepohlia (Xenococcus) kind.
Genome and other nucleotide sequences comprise modification and the synthetic nucleotide sequence that comes from this genoid group.Current described method is equally applicable to also undocumented nucleotide sequence---when they become can obtain the time, comprise characterize multicellular organism such as higher plant such as corn and paddy rice, Mammals for example, such as those of rodent (mouse, rat, rabbit etc.), pig, cow, bull, horse, primates, sheep and companion animals (, dog, cat etc.).In one embodiment, the cell separated from the mankind can be used for obtaining the donor gene group.The many of these are available at present.Nucleotide sequence and genome comprise those nucleotide sequence and the genome existed in simulating nature not.
In one embodiment, the selected genome that uses present method operation and other nucleotide sequences come from unmanageable biology or other have weak genetic system or with the host living beings biology of the unacceptable genetic system of comparing that---comprises some prokaryotic organism and other non-yeast bios---, such as common genetic technique wherein be poor efficiency those, such as double exchange homologous recombination and transposon mutant.For example, some bacterium living beings such as the mycoplasma kind in, these technology are poor efficiencys.Although the target for gene in mycoplasma mycoides LC adds and break (disruption), carried out changing event integrated plasmid DNA (Janis by single cross, C etc. 2005, Appl Environ Microbiol 71:2888-93), but this biology comprises the minute quantity selective marker, this has limited the quantity of the genetic modification that can carry out in single mycoplasma mycoides LC cell.
Interested is to have compound useful on manufacture and/or the biology of the potentiality that for example, work at extreme environment (, high temperature, high pressure etc.), and such as prokaryotic organism, it comprises those with weak genetic system.Exemplary biology comprises those that can be used for producing biofuel.Other exemplary biologies comprise those of experience photosynthetic process.Genome and biological can use described herein and methods known in the art to carry out genetic modification to generate new genome and the biology for the production of biofuel.For example, the gene that relates to photosynthesis and other metabolic processes can be modified to generate the biology that produces oily biological example fuel rather than glucose or another kind of carbon source.Therefore, the genome that this paper comprises is those genomes of being transformed biology with the ability that sunlight is become to biofuel with carbon dioxide conversion.A kind of so exemplary biology is proto green algae, and it is one of photosynthetic organism the abundantest on the earth, but has inefficient genetic system.
In one embodiment, carry out the method to modify and the modifying gene group, such as complete (complete) genome (for example, full cell, virus and organelle gene group) and a complete genomic part, it is included under at least one group of envrionment conditions, enough realize and/or maintain in cells survival (the smallest cell genome), host cell the existence (for example, minimum viral genome) of the biology that relies on host cell existence or the genetic stocks of organoid function (the smallest cell device genome) in host cell.On the one hand, genome is minimal genome or minimum replicator group.On the other hand, genome is included in the other nucleotide sequence be found in outside those in minimal genome or full genome.
Genome can be abiogenous or synthetic, such as the genetic modification genome, comprises the genome of modification and comprises the hybrid gene group from the nucleic acid more than a kind and/or portion gene group.
On the one hand, genome is cellular genome, and it comprises the nucleotide sequence that enough causes and/or maintain cells survival, and for example the coding copies, transcribes, translation, power generation, transportation, film and cytoplasm fraction produces and those of the needed molecule of cell fission.
On the other hand, genome is virus or organelle gene group.
In one embodiment, nucleotide sequence is the organelle nucleic acid sequence, for example, the organelle gene group, such as plastid, for example chloroplast(id) and Mitochondrial Genome Overview.The eukaryotic cell device, such as plastosome and chloroplast(id), be comprise cytoplasmic DNA, film is in conjunction with interval, it is considered to be in the residue of the endosymbiosis bacterium caught in their hosts.Usually, plastosome is natural to be seen in all eukaryotic cells, and chloroplast(id) sees in plant and algae with other plastids are natural.The size of plastom changes from 35 to 217kb, great majority 115 and 165kb between.The size of Mitochondrial Genome Overview does not alter a great deal between of the same race, and can from lower than 20kb to surpassing 350kb.The application's method allows to use nucleic acid and the genetic system engineered cells device genome in host cell in host cell.For example, organoid can use yeast plasmid and homologous recombination to be modified in yeast host.The method provided can be used for generating new plastosome or chloroplast gene group, for example, at eukaryotic cell, such as increasing power generation or metabolism in yeast and algae.
In another embodiment, nucleic acid is virus and bacteriophage nucleic acid, such as virus and phage genome.For example, virus and bacterial nucleic acid and genome provided method can be provided be modified and transform to generate the virus with therepic use.As another example, viral genome can be used method described herein to be cloned and operate.In medical use, virus has been used to gene therapy, vaccine and has been used as Trojan Horse (Trojan horses); But viral genome is for operating and Yan Taida in simple plasmid.Similarly, phage has been used as microbiotic decades; But the genome of T phage is too large, consequently can not easily process.
In another embodiment, passing through provided method modification, transformation and the nucleotide sequence generated is not genome.For example, nucleic acid comprises karyomit(e) and other nucleotide sequences.
Typically, nucleotide sequence and genome are large nucleotide sequences.On the one hand, the length of genome or other nucleotide sequences is at least or about 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1 megabase (MB), 1.1MB, 1.2MB, 1.3MB, 1.4MB, 1.5MB, 1.6MB, 1.7MB, 1.8MB, 1.9MB, 2MB, 2.1MB, 2.2MB, 2.3MB, 2.4MB, 2.5MB, 2.6MB, 2.7MB, 2.8MB, 2.9MB, 3MB, 3.1MB, 3.2MB, 3.3MB, 3.4MB, 3.5MB, 3.6MB, 3.7MB, 3.8MB, 3.9MB, 4MB, 4.5MB, 5MB, 6MB, 7MB, 8MB, 9MB, 10MB, 15MB or 20MB, or wherein any concrete numerical value or scope.The method provided also can be used for operation and the less nucleotide sequence of clone, such as, for example be less than those of about 100kb.
Ii. the propagation of donor gene group and other nucleic acid, separate and synthesize
Before shifting, nucleotide sequence can be bred and/or separate from cell or tissue in cell or tissue.The donor nuclei acid sequence can for example, separate or use clone, cell and plasmid technology and the system of knowing from donorcells or tissue (, from cell and the tissue of donor biology), is transformed into other cells and breeds therein.Nucleotide sequence in cell can be natural or synthetic, comprises partial synthesis.In some cases, after separating from cell or tissue, nucleotide sequence is such as being increased by PCR.
Donor nucleic acid also can be used chemosynthesis and assembly method chemosynthesis in vitro, and need not from any particular organization or cell, separate before therefore using in described method.Method for DNA and RNA chemosynthesis and nucleic acid assembling is known, and comprise that oligonucleotide is synthetic, assembling and polymerase chain reaction (PCR) and other amplification methods (such as, for example, rolling circle amplification, whole genome amplification), those that describe and describe in Gibson equals the U. S. application submitted on November 7th, 2008 number 12/247,126 such as this paper.For example, can, for example, from DNA (, passing through PCR) or from RNA, for example pass through reverse transcription, synthetic DNA.In nucleic acid is the synthetic gene group.For example, the synthetic gene group can be as described herein and the production described in Gibson equals the U. S. application submitted on November 7th, 2008 number 12/247,126.
Iii. nucleotide sequence, carrier-host system and culture condition
Nucleotide sequence can be from the separation of various sources, genetic modification, amplification and/or recombinant expressed/generation.The recombinant polypeptide generated from these nucleotide sequences can be separated or be cloned individually, and the activity of expectation is tested.Any recombinant expression system be can use, bacterium, Mammals, fungi, yeast, insect or vegetable cell expression system comprised.
Alternatively, nucleotide sequence can synthesize in vitro, such as the chemical synthesising technology by knowing, and/or obtain from commercial source, and optionally assembling, such as for large nucleic acids and genome, for example, at Gibson, equal described in the U. S. application submitted on November 7th, 2008 number 12/247,126.
Technology for the nucleotide sequence operation has been described fully in science and patent documentation, such as, for example subclone, label probe (for example, using random primer labelling, nick translation, the amplification of Klenow polysaccharase), order-checking, hybridization etc.
Obtain and operation is from genome sample clone for another process useful of the nucleotide sequence of implementing institute's supplying method, and if expectation, screening and cloning from the inset of for example genomic clone or cDNA clone and separate or amplification again.The source of the nucleotide sequence used in the system and method for describing comprises and is included in for example artificial mammalian chromosome (MAC), sees, for example U.S. Patent number: 5,721,118; 6,025,155; The human artificial chromosome, see, for example Rosenfeld (1997) Nat.Genet.15:333-335; Yeast artificial chromosome (YAC); Bacterial artificial chromosome (BAC); Pl artificial chromosome (seeing, for example Woon (1998) Genomics 50:306-316); Come from the carrier (PACs of Pl; See, for example Kern (1997) Biotechniques 23:120-124); Genome in cosmid, recombinant virus, phage or plasmid or cDNA library.
For separating of nucleotide sequence, as the method for genomic dna, know.As the technician be it is evident that, the method for separation depends on the type and size of separated sequence (one or more), biological type and the therefrom tissue of separation sequence (one or more) or the type of cell.For the method for separating genetic material from various biologies be know and can use in the present embodiment to separate donor nuclei acid sequence (genome).For example, the ordinary method of separating for DNA be know and can be according to size the method for providing, and available commercial availablely implement for separating of the test kits of nucleotide sequence in a large number; For example, commercial available test kit can be used for isolation of genomic DNA from cell.
Natural and synthetic nucleotide sequence can pass through amplified proliferation, for example copies.Amplification also can be used for the clone or the nucleotide sequence provided is provided.Therefore, the amplimer sequence pair of the nucleotide sequence provided for amplification is provided.Those skilled in the art can be any part or the total length design of amplification primers sequence pair of these sequences.Amplification also can be quantitatively in the quantity of sample amplifying nucleic acid sequence, such as the quantity of donor gene group in host cell.
Can select the also amplimer of appropriate design.Amplification method is also well known in the art and comprises, for example polymerase chain reaction---PCR, ligase chain reaction (LCR), transcription amplification (seeing, for example Kwoh (1989) Proc.Natl.Acad.Sci.USA 86:1173); And self-sustained sequence replication (seeing, for example Guatelli (1990) Proc.Natl.Acad.Si.USA 87:1874); Q β replicative enzyme amplification (seeing, for example Smith (1997) J.Clin.Microbiol.35:1477-1491); Automatically the technology (for example, NASBA, Cangene, Mississauga, Ontario) that Q-β replicative enzyme amplification test (seeing, for example Burg (1996) MoL Cell.Probes 10:257-271) and other RNA polymerase mediate; Also referring to Berger (1987) Methods Enzymol.152:307-316; Sambrook; Ausubel; U.S. Patent number: 4,683,195 and 4,683,202; And Sooknanan (1995) Biotechnology 13:563-564.
In one embodiment, nucleotide sequence at bacterial cell such as intestinal bacteria, intestinal bacteria DH10B[F for example --mcrA Δ (mrr-hsdRMS-mcrBC) ф 80dlacZ Δ M15 Δ lacX74 deoR recAl endAl araD139 Δ (ara, leu) 7697 galU galK λ -RpsL nupG] (Invitrogen)) in, use plasmid clone and propagation.In intestinal bacteria and other laboratory strains the method for clone and propagation nucleic acid and plasmid be know and can be combined with the method provided.In one embodiment, Bacillus coli cells is at substratum for example in Luria-Bertani (LB) broth culture or growth under 37 ℃ in LB agar.
But the fertile nuclei acid sequence, for example, in the mycoplasma cell.The mycoplasma cell can be the natural donor mycoplasma cell that comprises donor nucleic acid or donor nucleic acid for example the synthetic gene group be transformed into mycoplasma cell wherein.Exemplary mycoplasma kind comprises, for example, and mycoplasma capri subspecies capricolum (bacterial strain California Kid TM) (ATCC 27343) and mycoplasma mycoides subspecies mycoides (bacterial strain GM12) (Damassa etc., 1983), mycoplasma capri subspecies capricolum (mycoplasma capri), such as the wild-type mycoplasma capri and by as the embodiment be described below in CCATC-restriction enzyme allele inactivation in the wild-type mycoplasma capri is obtained mycoplasma capri mutant (mycoplasma capri-Δ RE).It is known making the method for these and other mycoplasma cells and other Growth of Cells.In one embodiment, the mycoplasma donor is comprising growth (Tully etc. 1977) in the liquid or solid SP4 substratum of 17% foetal calf serum (Invitrogen) under 37 ℃.
In order to be conducive to select the cell of the use currently known methods clone who comprises expectation and the nucleic acid of breeding, but engineered cells culture and pUC pUC.In one embodiment, bacterial cell, is for example grown in the substratum of the tetracycline of the tsiklomitsin of penbritin, the 5 μ g/ml of 50 μ g/ml or 125 μ g/ml being supplemented with microbiotic or other selective reagentss according to the nucleic acid resistant gene of plasmid for example be arranged in for clone and propagation such as intestinal bacteria.Similarly, the mycoplasma cell transformed with plasmid, donor gene group or other nucleic acid can for example be grown in the SP4 substratum at the substratum of the tetracycline of the tsiklomitsin that is supplemented with 5 μ g/ml or 8 μ g/ml.
Can use the method for knowing to detect the expression of expectation gene product in cell.For example, can be by the detectionofβ-galactosidaseactivity on the solid medium of the chloro-3-indyl-β of the bromo-4-of the 5-that comprises 150 μ g/ml-D-galactopyranoside (X-gal, Promega) by mycoplasma or other cell type bed boards.The technician can understand this paper and consider to use routine techniques to be used for other conditions and the method for expressing gene product.
A. separated nucleic acid sequence in the agarose inserted block
Nucleotide sequence can separate so that damage and minimize in the agarose inserted block.During conventional sepn process, large nucleotide sequence, such as scope from several kilobase to for example, to 10MB and larger DNA (, genome), can for example, by mechanical force (, dropper is drawn), be sheared, cause damage.Separating this class nucleotide sequence in agarose can make infringement minimize.In this embodiment, the cell that comprises donor dna is embedded in agarose and cracking.For in the agarose method of low melting-point agarose isolated genes group nucleotide sequence for example, be also know and can use commercial available test kit, such as CHEF mammalian genes group DNA inserted block (plug) test kit (catalog number (Cat.No.) 170-3591), CHEF bacterial genomes DNA inserted block test kit (catalog number (Cat.No.) 170-3592) and CHEF pastoris genomic dna inserted block test kit (catalog number (Cat.No.) 170-3593) are implemented, it is all from Bio-Rad Laboratories Hercules, CA.Using such test kit to prepare condition and the scheme that DNA can use supplier to recommend carries out.
Also can, according to the scheme of manufacturers's suggestion, use low melting-point agarose to separate donor nuclei acid sequence (for example, bacterium, mycoplasma, yeast or algae genome) with Bio-Rad CHEF mammalian genes group DNA inserted block test kit in the agarose inserted block.In one embodiment, cell suspension is in the substratum that there is no serum or PBS, and many cells (for example, prepare every mL agarose 5 * 10 7Or 5 * 10 8Individual) be centrifuged and be resuspended in the agarose for the treatment of half manufactured final volume.Simultaneously, prepare low melting-point agarose (for example, Bio-Rad2% CleanCut in sterilized water TMAgarose) and melt and balance to 50 ℃.Cell suspending liquid balance to identical temperature is also leniently mixed with agarose.Mixture is transferred in the inserted block mould and allows and solidify.In one embodiment, for molten born of the same parents, the mixture that interpolation comprises Proteinase K (for example, with 5ML Proteinase K reaction buffer, every mL inserted block comprises 100mM EDTA---pH8,0.2% Sodium desoxycholate, 1% sodium lauroyl sareosine and the Proteinase K of 1mg/mL), and be incubated overnight under 50 ℃.In another kind of embodiment, for molten born of the same parents, by the cell in agarose under 50 ℃, in 0.4M EDTA, 0.4%N-Sarkosyl L (N-lauroyl sarcosine), 2mg/mL Proteinase K, be incubated overnight two days during, then exchange buffering liquid processing for the second time the identical time under identical condition.
After molten born of the same parents, the agarose inserted block that comprises donor nucleic acid can be for 10mM, the Tris of pH 8.0 (EDTA that optionally comprises 1mM or 50mM) dialysis for example 1 hour.In one embodiment, after this dialysis, dialysis twice in each two hours in 10mM Tris, 50mM ETDA, 0.1mM PMSF, and again dialyse for storing in 10mMTris, 50mM EDTA.In another kind of embodiment, then for 10mM (6%) PEG6000 (United States Biochemical), 0.6M NaCl, dialyse a few hours, as Katsura etc., Electrophoresis 21,171 (Jan, 2000) are described, and then shift.In one embodiment, inserted block further melts 5 minutes under 65 ℃, then adds the TE of 65 ℃ of two volumes and leniently stirs, then 65 ℃ of lower incubations 5 minutes.
In some aspects, inserted block, before conversion enters host cell, is used for example β-gelase to melt and digest.In one embodiment, inserted block by twice of CHEF (Bio-Rad) electrophoresis (for example, for the first time on 1% pulsed field sepharose, with the switching time of 0.5 * TBE and 50-90 second, over 20 hours, for the second time on 1% low melting point gel, with 1 * TAE and 60-120 switching time second, over 24 hours), still stay complete genome to remove broken DNA, then, for aseptic 1 * TAE dialysis melting under 73 ℃, balance to 42 ℃ is also used β-gelase (New England Biolabs) digestion, for example 1.5 hours.
B. isolated cell device genome
The donor nuclei acid sequence can be the organelle gene group, the organelle gene group of for example separating from donorcells.This area is known for separating of the method for organelle gene group.From many manufacturerss, comprise Pierce (Rockford, 111.), Sigma-Aldrich (St.Louis, Mo.) and other the test kit for from cell isolated cell device are available.
Organelle gene group and other nucleic acid can, from various cell types, such as eucaryon and prokaryotic cell prokaryocyte, comprise in yeast cell, vegetable cell, algae and mammalian cell and separating.Should be appreciated that as known in the art, sepn process can be according to the Change of types of the biological and separated organoid of the cell of isolated cell device nucleic acid therefrom.The organelle nucleic acid sepn process can be included in the total genomic dna sample from nucleus DNA isolated cell device DNA, for example, according to molecular weight, by fractional separation or gel, separates.In some embodiments, before extracting DNA, organoid can be from total cell part purifying.
For from plant, separating the genomic method of chloroplast(id) (with other plastids), be known.Separate and usually by (1), to make that plastid and other organoids separate, (2) carry out chloroplast(id) cracking and (3) purification of nucleic acid.In one embodiment, sucrose or Percoll stepwise gradient be for obtaining chloroplast(id) from cell lysate, and it follows cleaved and for reclaiming DNA.In specific embodiment, by plant be placed on dark sentence reduce chloroplast(id) starch level, greenery in tap water rinsing and put into (10-100g) to dissociating buffer with the agitator homogenate in precooling, then filter by cheese cloth centrifugal.To precipitate (pellet) application of sample to one-level gradient (one-step gradient), its have 18mL 52% sucrose, be coated with 7mL 30% sucrose, optionally more saccharose gradients are to increase output, such as at least 6 saccharose gradients of 200g parent material.The centrifugal classification gradient, for example, under 4 ℃, 25,000rpm, 30-60 minute, and use the heavy caliber pipette to shift out the chloroplast(id) band from the 30-52% interface.Then as also centrifugal to remove fragment as description cracking chloroplast(id).Then for example use as described CsCl gradient purify DNA.DNAseI processes and can be used for improving the saccharose gradient method and destroy nucleus DNA.Alternatively, as well known in the art, high salt (for example, the NaCl of 1.25M) method can be used for separating and centrifugal without stepwise gradient.Can use the cell sorter (FACS) of fluorescence-activation to separate, sorting chloroplast(id) from plastosome and nucleus.
Comprise that for separating of Mitochondrial DNA (mtDNA) method of highly purified mtDNA is known, and can use with the separate mitochondria genome in conjunction with the method provided.Highly purified mtDNA can be used sucrose to cross pad (pad) gradient and the preparation of cesium chloride gradient from vertebrates or invertebrates tissue.Usually, from biology, if necessary, by homogenate, organize, then under the low speed repeated centrifugation to remove cell debris and nucleus, follow centrifugal under high speed and split throw out, (for example vertebrates and invertebrates embryo's brain, testis, ovary, liver, kidney, heart, skeletal muscle) is organized in preparation.Under 4 ℃, by 25,000rpm ultracentrifugation 1 hour, throw out is carried out to the sucrose stepwise gradient, the 10mL layer of crossing pad and 1M sucrose of the 1.5M sucrose that wherein gradient comprises 10mL in the TE damping fluid.For this process, throw out resuspension at the gradient higher slice in the TE damping fluid, then under 4 ℃, with the centrifugal 1-2 hour of 27,000rpm.Be collected in 1M and 1.5M sucrose is crossed the highly purified plastosome that shows as milky white colour band of the interface of pad by the Pasteur pipette, and be placed in pipe by high speed centrifugation (for example, under 4 ℃, 13,000rpm) further concentrated.
The then plastosome of cracking precipitation, for example, for example, by resuspension in the TE damping fluid and add sodium lauryl sulphate (SDS) (, every mL TE 0.3mL 10% SDS).After solution shows clarification, add saturated CsCl and in incubation mixture on ice 20 minutes, then with 12,000rpm centrifugal 10 minutes, retain supernatant liquor.For example, with CsCl-propidium iodide (PI) (or ethidium bromide) gradient (, every 8mL supernatant liquor 8g CsCl and 0.6mL 2mg/ml PI, mix and adjust density to 1.56g/mL), purify DNA.After centrifugal with 36,000rpm, top band comprises nucleus DNA, and following band comprises mtDNA, and it can be collected and optionally follow further CsCl gradient.Test kit for separating of mtDNA is commercial available.In one embodiment, use mtDNAExtractor CT test kit (Wako, Osaka Japan, catalog number (Cat.No.): 291-55301) from mammalian tissues, such as in muscle or liver organization, preparing Mitochondrial DNA.For example, for separating of fungi (, yeast) Mitochondrial DNA, the scheme of---as the superhelix cyclic DNA, it can not be released in cell homogenates usually effectively---is also known.In one embodiment, the CsCl density gradient centrifugation of the thick prepared product of DNA is carried out in situation about for example existing preferably, with the dyestuff (, DAPI or two benzimide (Bb)) that the DNA that is rich in AT is combined.In another kind of embodiment, mtDNA extracts from the plastosome separated, described plastosome is by making nucleus and cell debris precipitation in 20 minutes with the centrifugal lysate of 2000g, then on saccharose gradient, separation of supernatant is (for example, 2mL 60% sucrose, be coated with the sucrose of 4mL 50%, is coated with the sucrose of 4mL 44%, then with getting rid of the flat rotor, with centrifugal 90 minutes of 120,000g and from 44/55% interface, collect plastosome) separated.
D. donor gene group and nucleotide sequence are introduced to host cell
Is method and the nucleic acid of the donor nucleic acid introducing host cell for comprising the donor gene group in embodiment is provided, for example, at host cell, using the host cell machine to be modified.Host cell is to provide the heterologous host cell of expectation ability, and described ability does not exist usually in the cell in donor gene group source, the machine of for example recombinating.As above-mentioned, donor nucleic acid can from cell or tissue, separate then shift, in vitro chemosynthesis and/or assembling and/or by method in external or body from this class separation or synthetic nucleic acid replication.
Typically, at first by donor nucleic acid (it can be ring-type, linearizing or fracture) is connected with host's nucleic acid of host's carrier normally, the nucleic acid that comprises donor nucleic acid and host's carrier with generation, it can breed and modify in host cell.Donor nucleic acid and being connected of host's carrier can be carried out in the external or body at donorcells or host cell, donor nucleic acid (for example, donor gene group) is transferred to host cell.In one embodiment, host's carrier is transformed into donorcells, and it and donor gene group restructuring there then separates and has the genome (seeing, for example Fig. 2 A) that carrier inserts.In another kind of embodiment, donor gene group and host's carrier cotransformation respectively enter host cell, and donor and host's nucleic acid are recombinated in host cell.Before with the carrier corotation, dissolving host cell, the donor gene group can linearized (seeing, for example Fig. 2 B) or fracture (seeing, for example Fig. 2 C).
I. host cell
Host cell can be any host cell, and normally has the allos cell of the genetic system that is expected to be useful in modification of nucleic acids in laboratory, for example with donor biology or cell, compares the genetic system with improvement.The illustrative aspects of expectation genetic system is ability, selection definite and well-characterized and other mark group of supporting homologous recombination---to comprise double exchange homologous recombination and transposon mutant---and for cloning the ability of large nucleic acids.Also expecting that host cell has makes in host cell character compatible with donor nucleic acid during clone, propagation and modification of nucleic acids.
For example, can select specific host cell so that genotoxicity minimizes.Can select host/donor combination, so as in host cell, from the genetic expression of donor nucleic acid, not occur or with compare the genetic expression from donor nucleic acid in host cell reduce in donorcells.On the one hand, host and donor comprise different translations and/or transcribe signal and/or machine, such as yeast and bacterium living beings.On the other hand, one or more codons are translated into amino acid and are treated to terminator codon by the cell machine by donor.In one embodiment, donor translation cipher (for example, UAG) become amino acid (for example, tryptophane), and host cell is read identical codon for example, into terminator codon (, mycoplasma is to eukaryote).In these areas, donor gene group and other nucleic acid can in the host cell with expectation genetic system, retain, copy and modify and not (or minimum) express the gene product by the donor gene group coding.
Host cell can comprise any cell compatible with cloning donor gene group or nucleic acid.Therefore, for example, from the genome of algae, can be cloned into yeast operation so that how favourable characteristic to be provided when being reintroduced back to identical or different algae recipient cell.Reach the compatibility certain with system, these algae genes also can be operated and offer plant cell cultures.Similarly operation is feasible for vertebrates and invertebral zooblast.
A kind of preferred embodiment in, host cell is yeast cell.Yeast host comprises " pack horse kind ", and yeast saccharomyces cerevisiae and other yeast species are such as schizosaccharomyces pombe (Saccharomyces pombe), and it can be used for the genome that the clone is even larger.Due to the genetic manipulation set of tools of their uniquenesses, yeast host especially is applicable to operation donor gene group material.The natural ability of yeast cell and the research of decades have produced abundant for the set of tools at yeast operation DNA.These advantages are well known in the art.For example, yeast, the abundant genetic system with them, can be by the homologous recombination nucleotide sequence that assembles and reassembly, and this is the ability that the facile biology of many appearances does not possess.Yeast cell can be used for the larger DNA that the clone can not be cloned into other biological, for example, and whole cell, organoid and viral genome.Therefore, a kind of embodiment of institute's described method utilizes the huge ability of yeast heredity, by using yeast as the genome for operating unmanageable and other biological and the host cell of synthetic gene group, advances synthetic biology and synthetic gene group.
VL6-48 (ATCC MYA-3666TM)), W303a bacterial strain and restructuring-defect yeast strain exemplary yeast host cell is yeast strain VL6-48N, is the parental strain of high transformation efficiency exploitation:, such as RAD54 gene-defect bacterial strain, VL6-48-Δ 54G (MAT α his3-Δ 200 trp1-Δ 1 ura3-52 lys2 ade2-101 met14 rad54-Δ 1::kanMX), it can reduce the generation of multiple recombination event in yeast artificial chromosome (YAC).
Have many groups of checkings, that confirm and reliably for selecting and the counter selective marker of selecting yeast mutants, its make in yeast host cell, carry out many rounds for example the seamless nucleic acid of unlimited iteration round change and become possibility.Therefore, yeast can be used for introducing many different genetic modifications, comprises that mononucleotide changes (for example, insertion, disappearance, sudden change), modifies the target nucleic acid part and also builds brand-new karyomit(e) with zone.Continuous modification to other unmanageable genomes or other large nucleic acids clone copy can in extremely rapid succession be carried out in yeast.The mating ability of yeast is favourable for modifying factor group and other large nucleic acids.When yeast activated between mating season, yeast restructuring machine can be used for generating library, for example, and the combinatorial library that comprises colone genome or nucleic acid variant.
For example, for several different bacteriums built yeast artificial chromosome (YAC) library (Azevedo etc., PNAS USA 90,6047 (1993); Heuer etc., Electrophoresis 19,486 (1998); Kuspa etc., PNAS USA 86,8917 (1989)).Large procaryotic DNA fragment can be used general genetic code to clone in yeast.Poisonous genetic expression is not the obstacle of clone's donor nucleic acid in yeast usually.For example, the genomic research indication of bacterium and Archimycetes is because eukaryote and these bacteriums are used different protein expression machines, so the albumen of expressing from colone genome has harm risk seldom to yeast host.Yeast transcription (Kozak, Gene 234,187 (1999)) and the translation (Kornberg, Trends Cell Biol 9, M46 (1999)) signal from bacterium transcribe and translate different.In fact, most of prokaryotic gene is not probably expressed in yeast.Do not limit obstacle (Belfort and Roberts, Nucleic Acids Res 25,3379 (1997) in yeast.If obstacle is arranged, it may be to copy obstacle so, rather than genetic expression obstacle (Stinchcomb etc., PNAS USA 77,4559 (1980)).Genotoxicity is minimized, this be because eukaryote such as different from prokaryotic organism are regulated in the genetic expression in yeast.And mycoplasma accesses to your password sub-UGA as tryptophane rather than as the translation termination signal.Therefore, most of mycoplasma base because of, if express, will in yeast, produce the protein of brachymemma.This has been avoided the possibility of poisonous gene product to a great extent.
Can from the donor biology, obtain the donor of its natural form, and with yeast vector, donor is modified, then transform and enter yeast, or assemble together with yeast vector from natural or synthetic fragment, then transform and enter yeast cell, or donor while cotransformation together with yeast vector enters yeast cell.By these genomes being transferred to the new biology of creation in compatible recipient cell, described these genomes are optionally by the expectation operation.Therefore, a kind of embodiment provides applicable technology first, be used for: genome is transferred to yeast host cell, in host cell, the modifying factor group retains their stability and integrity simultaneously, and return to the more closely recipient cell of similar initial donor from the genome that yeast host cell shifts clone and operation, thereby manufacture the previous non-existent and/or biology that can't manufacture by available genetic modification and their initial cell of clone's instrument genetic manipulation.
Ii. host's carrier
Typically, use host's carrier that donor nucleic acid is transformed into to host cell and breeds therein.Therefore, host cell generally comprises or supports to introduce host's carrier, for shift, retain and modify donor nucleic acid at host cell.In one embodiment, host's carrier comprises nucleotide sequence to be conducive to from donorcells transfer donator nucleic acid to host cell and recipient cell and other cells, such as for clone and propagation bacterial cell (for example, intestinal bacteria), for example, such as three shuttle vectorss of describing in this paper embodiment (seeing, Fig. 3).
On the one hand, carrier is included in one or more expectation cell types and promotes carrier to copy required any nucleic acid (for example, replication origin) and for selection and/or the resistance marker of different cell types.
Resistance marker is known.The technician can determine suitable resistance marker for different hosts/donor combination.In some cases, non-clinical relevant mark is used in expectation.In other cases, the character of donor, host and/or recipient cell is depended in the selection of resistance marker.For example, the microbiotic of targeted cells wall may be used in mycoplasma and other lack the biology of cell walls.One of resistance marker is the coding antibiotics resistance; such as the gene of penbritin, kantlex and tetracyclin resistance, such as tetracycline resistance protein (TetM) and paraxin acyltransferase (CAT), aminoglycoside resistance protein (aacA/aphD) and its combination.For example, the tet-resistance marker can be used for bacterium such as in mycoplasma, and wherein tsiklomitsin has effective effect and the low-level spontaneous resistance of its displaying.Also can use the gene of giving the tetracycline resistance, for example, for clone and modification mycoplasma nucleic acid and use mycoplasma cell.
Tetracycline be the simulation aminoacylation tRNA 3 '-end and be attached to the growth protein chain carboxyl terminal to stop the synthetic microbiotic of albumen.Because tetracycline is enlisted by the rRNA recognition component that in cell, all various tRNA are used, and is impossible so can obtain spontaneous antibiotics resistance by simple point mutation, it can occur with other marks in some cases.Tetracycline is to hold facilely, relatively cheap, does not use clinically and it is in prokaryotic organism and eukaryote effective translational inhibitor in the two.There do not is the known resistance based on rRNA.
Developed codon optimized box in 5 kinds of different mycoplasma kinds, to give the tetracycline resistance, and can work in intestinal bacteria, made it that function be arranged in shuttle vectors.In order to make this box, use overlapping oligonucleotide to synthesize 597bp tetracycline N-acetyl-transferase 85 genes (PAC), as Smith etc., PNASUSA 100:15440-5 (2003) describes.In brief, the oligonucleotide (in order to express in mycoplasma genitalium) of 5 ' phosphorylation of two chains of coding password optimization version is ordered from IDT (Coralville, IA).Few nucleic acid is 48 bases, and 88 is long, has 24 bases overlapping.Mix the few nucleic acid of Lian He downstream, upstream chain, be heated to 95 ℃ and Slow cooling so that overlapping annealing.Connect (ligate) and react 12 hours, and be used as the template of PCR.Pcr amplification is cloned into pGEM-3Zf (+) (Promega, Madison, Wisconsin) and checks order to identify correct pac clone.Then, the PAC gene optimized is controlled to lower clone and for replacing at the tetM of Mini-Tn4001tet derivative gene in spiroplasma citri spiralin promotor (Ps), and the terM gene in pMycol (Lartigue etc. (2003), Nucleic Acids Res 31:6610-8).New plasmid (Mini-Tn4001PsPuro) can be used for transforming mycoplasma genitalium, Mycoplasma gallisepticum and mycoplasma pneumoniae, and pMycol derivative (pMycoPuro) can be used for transforming mycoplasma mycoides LC and mycoplasma capri.
Carrier also comprises and allows the nucleic acid that carrier is connected with donor nucleic acid.In one embodiment, the homology zone that host's carrier comprises donor gene group or nucleic acid, such as linear carrier 3 ' and 5 ' end the homology zone, its with donor nucleic acid in the adjacent domain homology, with by homologous recombination, promote the connection.In another kind of embodiment, the nucleic acid that host's carrier comprises coding transposase and/or inverted repeat, connect into such as inserting such as the donor nucleic acid in donorcells being conducive to.The host is carried and can additionally be comprised restriction enzyme recognition site and nucleic acid and support copy and separate to neutralize at host cell in other cells.
On the one hand, the yeast host carrier comprises replication origin (for example, from the height of pUC19, copying initial point); One or more resistance markers and/or selective marker (for example, antibiotics resistance gene and selectable host cell (for example, yeast) mark), such as the mark for selecting in host cell, donorcells and recipient cell.Exemplary resistance/selective marker is antibiotics resistance gene (for example, penbritin-resistant gene, kalamycin resistance gene and other antibiotics resistance genes of knowing) and other antibiotics resistance genes; Selectable yeast or other host cell marks, (for example, HIS3) and/or selective marker; Be conducive to insert the nucleic acid of donor nucleic acid, for example transposase and inverted repeat, such as enter the mycoplasma genome for swivel base; Be supported in host cell and copy and the nucleic acid separated, such as autonomously replicating sequence (ARS), centromeric sequence (CEN).In one embodiment, carrier comprises telomeric sequence.
Exemplary carrier comprises the carrier of yeast, and it comprises yeast centromeric plasmid, and yeast artificial chromosome (YAC) carrier for example, such as EXAMPLE l A (i) below (a) describes and diagrammatic pmycYACTn in Fig. 3 A; With the miniTn-Puro-JCVI-1.7 carrier that demonstration builds in Fig. 3 B.The feature of pmycYACTn carrier comprises: (i) height from pUC19 copies initial point and amicillin resistance mark, (ii) IS256, (iii) terM and lacZ mark for breeding intestinal bacteria, the two is from spiralin promotor (16,17) express, for intestinal bacteria and mycoplasma, being selected and screened, (iv) ARS and CEN, for at yeast, being copied and separated, and as the HIS3 of selectable yeast selectable marker.The miniTn-Puro-JCVI-1.7 carrier is different as follows from pmycYACTn's: (i) it does not comprise lacZ and replaces terM and (ii) it comprises bacterial artificial chromosome (BAC) carrier with the tetracycline resistance marker, for the clone possible intestinal bacteria.
Iii. Strategies For The Cloning: connect host and donor nucleic acid and also donor gene group and nucleic acid are transferred to host cell
In the transfer method provided, donor gene group or other nucleic acid and host's nucleic acid, normally host's carrier connects, the nucleic acid that comprises donor nucleic acid and host's nucleic acid, can in host cell, breed and operate with generation.Can use many methods of extracting in the cloning process of knowing to be connected host and donor nucleic acid.Describe in more detail below three ordinary methods.
As top institute record, yeast cell is exemplary host cell.Large DNA molecular stably has been cloned into yeast by adding yeast kinetochore (CEN), and described kinetochore allows molecule to separate together with yeast chromosomal.Such molecule is cloned by adding telomere to end with the wire form, and also as ring-type, clones.Because bacterial genomes is the cardinal principle ring-type, and encircles and easily separate with the wire yeast chromosomal, so can be favourable with ring-type cloning bacteria genome.
For connecting the donor gene group and thering is the first method of the nucleic acid of host's carrier, the donor gene group is connected to host's carrier in donorcells or other cell types similar to donor, then separate the nucleic acid that comprises donor gene group and host's carrier, and shift and enter host cell subsequently.Embodiment is illustrated in Fig. 1.For example, the method can be used for the metastatic gene group and other large nucleic acids enter host cell.In one embodiment, the nucleic acid that host's carrier comprises inverted repeat and/or coding transposase, to be conducive to inserting donor gene group or other nucleic acid in cell.
Provide advantage at donorcells or for example, with the similar cell of donor (, same genus is not of the same race) middle connection donor and host's nucleic acid.For example, it allows to select carrier to insert (for example, along the carrier insertion point of donor length nucleic acid), and it can maybe can not slacken or otherwise affect donorcells existence.For example, but the method needs donor or similar cell easily to introduce exogenous nucleic acid (, transforming) really, for example, so that this carrier can be integrated into donor nucleic acid, genome.For many methods of nucleic acid being introduced to the various kinds of cell type, know.In one embodiment, described in the embodiment 1A, the yeast host carrier under existing situation, PEG is transformed into to bacterial cell as following.For example, according to the resistance in host's carrier or other selective markers, select the donorcells that comprises the host's carrier that is integrated into donor nucleic acid.Nucleic acid can separate from donorcells, such as above-mentioned in the agarose inserted block, for confirming host's carrier, inserts, and for example passes through PCR or southern blotting technique as described herein.On the one hand, before being transferred to host cell, the nucleic acid that comprises donor gene group and host's carrier is transferred to recipient cell to confirm this nucleic acid and can be transferred and compatible with specific recipient cell.For example, from a kind of bacterial species metastatic gene group DNA to can be as at Lartigue etc. another, in Science 317,632 (2007), describe and as EXAMPLE l A (ii) describes in (b) below carrying out.
In Fig. 2 A, in diagrammatic embodiment, in bacterial cell, linear yeast host carrier is connected with the ring-type bacterial genomes.The gained circular nucleic acid for example separates and is transformed into yeast host cell in agarose inserted block described above.The embodiment of this process describes in embodiment 1A below.
In embodiment, be illustrated in the second method in Fig. 2 B, donor gene group together with host's carrier or dividually corotation dissolves host cell, and they for example connect by homologous recombination in host cell subsequently.The method has superiority aspect simplicity, has minimum sample preparation and number of steps.Typically, as shown in Fig. 2 B, carrier inserts donor gene group or nucleic acid by homologous recombination.
In Fig. 2 B diagrammatic embodiment, linear yeast host carrier is together with ring-type bacterial genomes synthetic genome or from donorcells for example---for example as above-mentioned the agarose inserted block---, and the genome corotation of separation dissolves yeast host cell.Typically, host's carrier comprises the zone (one or more) with donor gene group or nucleic acid homology.In the embodiment that is presented at Fig. 2 B, linear yeast vector comprises the homeologous zone with bacterial genomes at each end.In one embodiment, as shown in Fig. 2 B, bacterial genomes is cut with restriction enzyme, and then near the cutting zone with host's carrier homology of this restriction enzyme transforms and enter host cell.This process generates double-strand break near the insertion point of carrier, and this has improved the efficiency that connects host's carrier and donor gene group (by carrier is inserted to genome) in host cell greatly.Typically, selectional restriction enzyme recognition site in donor gene group or other nucleic acid, its integrity with maintainer gene group or nucleic acid after this site insertion vector is compatible.The embodiment of this process describes in example I B below.
The embodiment of improved the third method of second method is illustrated in Fig. 2 C.By by a plurality of overlapping nucleic acid fragments,---it is donor gene group or nucleic acid fragment---dissolves host cell together with host's carrier corotation and carry out the method.In other words, the homologue that each fragment comprises donor gene group or nucleic acid region and along the homology overlapping region of donor gene group or length nucleic acid.Once be transformed into host cell, fragment and carrier restructuring, for example, by the homologous recombination in homology zone.
In Fig. 2 C diagrammatic embodiment, the overlapping fragments of ring-type bacterium donor gene group dissolves yeast host cell together with linear yeast vector corotation.Again, yeast vector comprises the homeologous zone with bacterial genomes at its end.Once donor gene group fragment and yeast host carrier are introduced to host cell, fragment and carrier restructuring, thus connect donor gene group and host's carrier.Embodiment describes in embodiment 1C below.
In some embodiments, for example, for example, with (, first method) after host's carrier is connected or before (, the second and the third method), as above-mentioned from donorcells or similarly cell, separate donor nucleic acid.For example, comprising genomic large donor nucleic acid can separate with other cells from donor in the agarose inserted block, as mentioned above.Isolated or synthesized and the assembling after, donor nucleic acid is transformed into to host cell.When host's carrier before is not connected with donor nucleic acid, it can use identical method for transformation, simultaneously or in succession be transformed into host cell with any order.
Method for transformation is well known in the art and will changes according to host cell.In one embodiment, when host cell is yeast host cell, prepared by the yeast spheroplast from yeast host cell, for example as described below, and nucleic acid is by mixing and transformed with spheroplast.In some cases, under existing situation, PEG transformed.For example, donor nucleic acid and/or host's carrier can with spheroplast incubation 10 minutes at room temperature, then add 800 μ L PEG 8000, and by being inverted gentle the mixing, and incubation another 10 minutes at room temperature.
In one embodiment, spheroplast preparation and transform as by Kouprina and Larionov, Nat Protoc3,371 (2008) carry out with describing.Growth of Cells OD extremely can change.Use these methods, before transforming, the unicellular colony inoculation of yeast host for the yeast culture base, and 30 ℃ of lower overnight growth, with forced oscillation to guarantee good ventilation, until reach suitable OD660.Sample can be centrifuged and be resuspended in Sorbitol Powder by vortex, and centrifugal and resuspension, for example, at SPE solution (1M Sorbitol Powder, 0.01M sodium phosphate, 0.01MNa 2EDTA, pH 7.5) in.For example use Zymolase TMRemove yeast cells wall.By relatively in sorbitol solution, to the optical density(OD) of the cell suspending liquid in 2% SDS solution (wherein spheroplast is cleaved), assessing the level of spheroplast.Spheroplast can be centrifuged and be resuspended in the 1M Sorbitol Powder by shaking as mild as a dove, and in STC solution (1M Sorbitol Powder, 0.01M Tris-HCl, 0.01M CaCl 2, pH7.5) rinsing and resuspension.By mixing nucleic acid and spheroplast, transformed, as mentioned above, optionally under there is situation in PEG.
After conversion, usually carry out the cell of chosen process to select donor nucleic acid and host's carrier successfully to be transformed into.For example, in said process, after conversion, spheroplast can be centrifuged and be resuspended in SOS solution, and in 40 minutes nonoscillatory of 30 ℃ of lower incubations.Spheroplast can be placed on the selection substratum, and such as the SORB-TOP-His of melting described herein selects in substratum, and 50 ℃ of lower balances, and bed board comprising on the flat board of selecting substratum, and for example under 30 ℃, grows, until transformant is visible.
Iv. from host cell, separate and analysis donor nucleic acid
Pass through provided modifying method in host cell, modify before and afterwards, the method provide is provided, separable and analyze the donor nucleic acid that is transformed into host cell.With the same with other cellular segregation from donor, according to cell type, separation method can change.In certain embodiments, can from the nucleic acid samples separated, remove or reduce natural host nucleic acid, for example, to separate or enrichment donor nucleic acid.But this process can be removed the karyomit(e) host DNA or be undertaken by the restriction enzyme digestion that use digests host's nucleic acid indigestion donor nucleic acid by prerunning.
In one embodiment, for example, use " cerevisiae dna that the prepares the agarose embedding " scheme in Bio-Rad CHEF-DR III handbook, and as at this paper embodiment described in, separation donor nucleic acid in the agarose inserted block.In certain embodiments, under constant voltage, prerunning comprises from the agarose inserted block of host cell DNA several hours to remove the yeast host chromosomal DNA.On the one hand, removing host DNA can be undertaken by the DNA at first digested in inserted block with AsiSI, Fsel and RsrII or other enzymes, described other enzyme cutting yeast chromosomals still do not have recognition site on donor nucleic acid, such as mycoplasma genitalium or mycoplasma mycoides LC.
The analysis of donor nucleic acid can be undertaken by any many methods for analyzing DNA of knowing.Typically, expectation is carried out method to confirm size and/or the sequence of nucleic acid, and correct insertion and the orientation of carrier and other nucleic acid, comprises and confirms any modification.In one embodiment, the DNA separated is by heating and/or restriction digestion experience linearizing effect, then by gel electrophoresis, such as reversing electric field (Bio-Rad FIGE Mapper) or pulsed field (Bio-Rad CHEF-DR II or III system) electrophoretic separation.
For example, can pass through PCR, such as multiplex PCR assay, the primer of design along the length of the donor nucleic acid of expectation donor nucleic acid or modification in conjunction with a plurality of zones.Typically, also design primer recognition of host carrier.In other embodiments, visual by the size that makes isolating nucleic acid on gel, or digest and carry out southern blotting technique or other hybridizing methods by restriction, for example, as Gibson etc., Science 319,1215 (2008) described analyses.The MPCR of separation donor gene group and the specific embodiment that southern blotting technique is analyzed have been described in an embodiment.The improvement of analytical procedure will be apparent for the technician.Sequence measurement is known, and also can be used for analyzing the donor nucleic acid that is transferred to host cell and breeds therein.
V. generate the host cell that comprises a plurality of donor gene groups
In one embodiment, by a plurality of donor nucleic acid, such as a plurality of genomes from different donors are introduced single host cell.On the one hand, the host cell that comprises the nucleic acid shifted from the different donors hybridization that the host cell that comprises a donor gene group or other donor nucleic acid is such from another, generate the host cell that comprises two kinds of nucleic acid.For example, comprise from two kinds of donor gene groups of different donors such as from the genomic diploid yeast bacterial strain of different types of two mycoplasmas, can be by two different haploid strains of hybridization-each carries a kind of donor gene group---generate.Can use the method for knowing to carry out the haploid yeast strain hybrid.The selective marker of many uniquenesses can be used on separately in haploid strains, to allow selection after hybridization, comprises two kinds of genomic cells.For example, HIS3 and TRP mark can be introduced respectively in two different haploid cells that carry different donor gene groups, then on the substratum that lacks Histidine and tryptophane, be selected diploid cell, described at this paper embodiment.
E. modify the donor gene group in host cell
In provided embodiment, be also method and the nucleic acid of modifying donor gene group and other nucleic acid in host cell.In one embodiment, by one or more target constructs are introduced in donor nucleic acid and are carried out described method.This construct comprises and the part of donor nuclei acid homology, resistant gene, selectable mark, codase nucleic acid, restriction site and/or other nucleic acid used in clone and homologous recombination such as restriction enzyme.Typically, this construct is introduced to the host cell that comprises donor nucleic acid.
For designing this construct, select the target region of donor nucleic acid (for example, donor gene group) to be modified.Method there is no need to modify each residue of target region.For example, can be modified at one or more target parts or the target position in target region.Modification is included in other modifications of insertion in target region, disappearance, sudden change, replacement and/or one or more Nucleotide.On the one hand, donor nucleic acid is by seamless modification (seamlessly modified).
Typically, target region or its part are at first with comprising the nucleic acid construct thing displacement of mark such as anti-selective marker.Then, by the disappearance mark or with another nucleotides sequence column permutation, remove mark from nucleic acid.On the one hand, by introducing have with mark part near or second nucleic acid construct alternative label and peripheral part of part (one or more) homology of contiguous target region.This second construct needn't be less than 100% homology with the part (one or more) of target region.For example, with the part of target region, compare, this construct can comprise one or more sudden changes, disappearance or insertion, once whereby with this construct displacement, target region is modified.The method usually comprises one or more homologous recombination steps.
On the one hand, for example, by introducing fracture (break) (, double-strand break) in the nucleotide sequence of the donor nucleic acid comprising the markd construct of tool, be convenient to remove mark.In near for example contiguous target region or introducing fracture in target region target region.Usually, identify and cut the enzyme of expectation nucleotide sequence by abduction delivering, such as endonuclease generates fracture.Usually, enzyme is by target region or mix the nucleic acid encoding in the construct of target region.
On the one hand, by nucleotide sequence being introduced to donor nucleic acid, help to remove mark, the flank that is inserted in target region or its part of nucleotide sequence generates the Tandem repeat territory whereby.Usually, nucleotide sequence is included as the part of target construct.
In one embodiment, method comprise introduce fracture and introduce generate the Tandem repeat territory sequence the two.On the one hand, method is that the series connection of bind nucleic acid restriction endonuclease cutting repeats (TREC) method, and the double-strand break wherein generated by the abduction delivering enzyme and series connection are recycled and reused for and help recombination event and avoid infringement and unwanted sudden change.
With the conventional method available with other, compare, the method provided provides advantage, in particular for modification donor nucleic acid in not of the same race, genus and purpose host.On the one hand, can be from the donor nucleic acid of the donor biology with weak genetic system, the host with abundant genetic system, such as being modified in yeast host cell, for example, by the homologous recombination method.For example, the nucleic acid fragment that length is hundreds of kb can be used method and the genetic tool of standard---yeast artificial chromosome (YAC) who comprises linear and annular form---known to clone and operate in yeast (yeast saccharomyces cerevisiae) host cell.The donor nucleic acid of modifying is transferred to the recipient cell that comprises initial cell and cell not of the same race and be can be used for for example functional study and the generation modifying factor product of gene and gene regulating.The method provided is used in host cell successfully modifies the donor gene group, to transform and to modify the genome from difficult biology in heredity.
As following, discussed, modifying method can be used for producing genome, biology and by biogenic commercial useful gene product, such as for generation of vaccine, medicine, bioprotein and chemical, biofuel and protein for treatment agent (protein therapeutics) such as enzyme and antibody.In one embodiment, modify the donor gene group to produce new immune component to cause immunne response, such as live virus and other immunogens.In another kind of embodiment, for example, by the encode DNA of enzyme of participation oil route of synthesis of introducing, for example by use, produce those pathways metabolism gene substitution pathways metabolism genes of biofuel, modify the donor gene group for the production of biofuel.In one embodiment, modify donor gene group (for example, the donor gene group of photosynthetic bacteria), once so that transfer enters recipient cell, recipient cell generation biofuel rather than normal photosynthate are such as glucose.Other purposes are discussed below this paper.Therefore, the method provided can be used for for example in yeast host cell, directly transforming or the synthetic bacterial genomes of redesign in body.
The modifying method provided comprises for overcoming aspect donor not of the same race and the uncompatibility problem between host living beings, itself otherwise can cause unstable and the unwanted sudden change of the donor nucleic acid operated in host cell not of the same race.For example, when donor nucleic acid is introduced to host cell such as the donor gene group, donor nucleic acid usually can not contribute to the existence of host cell, or can not contribute to the host's that separates with the independent selective marker (one or more) existed in cloning vector existence.When donor and host are dissimilar biologies, during such as different orders or boundary, for example when donor be that prokaryotic organism and host are eukaryotes during such as yeast, this is especially correct.For example, in the research of describing as embodiment below 4, discuss, in yeast, as the mycoplasma genitalium genome of ring-type YAC (thering is histidine mark) propagation, except Histidine prototroph, its host is not had to supplementing on function.Any disappearance of bacterial genomes and rearrangement may be neutral for yeast host.
Use available method, do not survive because host cell does not rely on the integrity of donor nuclei acid, so, when donor nucleic acid operates in host cell, have the excessive risk of unwanted sudden change and infringement for donor nucleic acid.The method provided overcomes these problems and is used in propagation and modification donor gene group in host cell, makes the risk minimization of unwanted sudden change in the donor gene group simultaneously.The method provided can accurately efficiently modify be cloned into yeast host cell the donor gene group such as bacterial genomes.
The method provided also is used in seamless modification donor nucleic acid in host cell, is included in sudden change in the target region of donor nucleic acid, disappearance and/or inserts Nucleotide, does not wherein add or removes unwanted other nucleotide sequence.
I. anti-selective marker
Usually, the first step of method comprises anti-selective marker introducing donor nucleic acid.Usually, this mark inserts by homologous recombination, and a part of whereby target region is replaced by anti-selective marker.Anti-selective marker is favourable, because the existence of mark and do not exist the two all can be selected.Mark exists available one group of growth conditions to select, and does not exist available a different set of growth conditions to select.An exemplary anti-selective marker of knowing is the URA3 yeast genes.In yeast host, exist the URA3 gene to allow it to grow lacking on the substratum of uridylic.Therefore, can be by uridylic-substratum, growing and selected with the displacement donor nucleic acid target region of this mark success.In contrast, there do not is the URA3 gene, such as after by another homologous recombination event displacement, can be selected by the anti-selection on the substratum thering is 5-fluororotic acid (5-FOA).
For example, genetic marker URA3 can for example be integrated into the target region in the donor nucleic acid that is cloned into host cell by homologous recombination.By lacking growth selective marker integration on the substratum of uridylic.For example,, by disappearance or for example, for example, with another nucleotides sequence column permutation---in second takes turns homologous recombination, by anti-selection, on 5-FOA, selective marker is removed.
They adopt the method for anti-selective marker to expect, because can be used for seamless modification.In addition, replace or remove anti-selective marker and recover auxotroph (for example, relying on uridylic), so that host cell can be used identical method to be modified in lower whorl is modified.
Method is used in yeast host cell introduces and replaces anti-selective marker.For example, currently known methods is introduced the URA3 mark by first round homologous recombination, and with the second homologous recombination replaced mark of taking turns.The example of this method is described in embodiment 4A below, wherein use this ordinary method that relates to two serial homology recombination event, carry out site-specific mutagenesis, to proofread and correct visible list base cytidine disappearance (309,388) in synthetic genomic CDS 139 locus of mycoplasma genitalium of the donor retained in yeast.As described in this embodiment, the yeast host that comprises mutant bacteria donor gene group is with comprising the URA3 mark and transforming the target region that displacement comprises single base deletion CDS 139 locus with the box of the homeologous 50bp terminal portions of target region.Second takes turns the construct that conversion will comprise not mutated body DNA sequence dna draws back the same gene seat, replaces this mark.
Adopt the ordinary method of anti-selective marker, be restricted with regard to them with regard to the ability of effectively modifying some donor nucleic acid in some host cell.For example, modifying the donor gene group in the host cell of these methods for the integrity existence not relying on the donor gene group may be poor efficiency.When host cell does not rely on the integrity of donor gene group, during modifying, many spontaneous deletions can occur.These disappearances usually cause the loss of anti-selective marker, and therefore selected.The method provided has overcome this problem and compared with ordinary method provides the efficiency increased.
Ii. fracture is expressed and introduced to inducible enzyme
In one embodiment, after introducing selective marker, for example, introduce fracture in (, contiguous) or target region near target region, such as double-strand break (DSB).Usually, identify and cut by abduction delivering and be arranged near target region or the enzyme of the nucleotide sequence of target region, such as endonuclease, for example I-SceI carries out this process.Usually, this enzyme is endonuclease or other enzymes that generate double-strand break.It is reported, introducing the double-strand break homologous recombination efficiency near the homologous recombination site increases about 20 times (Leem etc., Nucleic Acids Res 31, e29 (2003)).Therefore, carry out introducing double-strand break near target region, increase the efficiency of modifying method and reduce unwanted background sudden change.
In typical embodiment, the target construct that comprises selective marker also is included in the gene that inducible promoter is controlled lower codase.Usually, this construct also comprises the recognition sequence of enzyme.This construct is introduced donor nucleic acid in host cell.Growth by host cell under the specified conditions of inducing self-induction type promoter expression, the expression of inducible enzyme.In one embodiment, promotor is the GAL1 promotor, can by comprise semi-lactosi as the substratum of sole carbon source on growth inducing from its expression.
Available method comprises in order to improve the purpose of the efficiency of the modification based on restructuring in yeast, induces the introducing double-strand break.A kind of such method, Delitto perfetto(fixed-point mutation method), being described in Storici etc., Nat Biotechnol, in 19,773-776 (2001).The example of the method is described in embodiment 4B (i) below.The method (stimulates the performance (Storici etc., PNAS USA, 100,14994-99 (2003)) of several orders of magnitude of stimulate) recombinating based on introduce double-strand break (DSB) raising in target nucleic acid.In embodiment 4B (i), use Dilletto perfetto to attempt to proofread and correct identical single base deletion in mycoplasma genitalium donor gene group CDS 139 locus.Process is illustrated in Figure 10 A.
This paper shows that inducing introducing DSB in conjunction with conventional recombination method in yeast is restricted for modifying some donor nucleic acid.See following embodiment 4B (i).This restriction is the high background (background) due to the spontaneous loss of negative (instead) selective marker.The method provided has been improved efficiency and has been reduced unwanted background sudden change (for example, spontaneous deletion).
Iii. series connection repeats
In one embodiment, the flank by the zone comprising mark exists the Tandem repeat territory to contribute to remove selective marker by homologous recombination.On the one hand, for the target construct of introducing mark, also comprise nucleotide sequence, the flank that target region or its comprise the part of inserting mark that is introduced in of described nucleotide sequence produces the Tandem repeat territory.Construct inserts upstream or the downstream of target region or its part at first, and comprises and have respectively and target region or the downstream of part or the nucleic acid moiety of upstream portion homology.Insert this part near homology part in target nucleic acid, generate series connection and repeat.
In one embodiment, insert construct and generate the 5 ' part had with the 3 ' homeologous mark of target region in target nucleic acid.In another kind of embodiment, insert construct and generate the 3 ' part had with the 5 ' homeologous mark of target region in target nucleic acid.Therefore, once introduce, the donor nucleic acid of modification (for example, the donor gene group of modification) comprises tandem repetitive sequence at the nucleic acid flank that comprises selective marker.
The existence that series connection repeats order promotes the homologous recombination between two sequences, for example, and for removing the part of the construct that comprises anti-selective marker.This method be know and based on by homologous recombination (HR) between two tandem repetitive sequences, accurately excising nucleic acid fragment.An example, be commonly referred to " series connection repeats to give prominence to " method, is described in Akada, R. etc., and Yeast, in 23,399-405 (2006).The example of this method is described in following embodiment 4B (ii), for lacking the zone of mycoplasma genitalium donor gene group CDS 139 locus.Process is illustrated in Figure 10 B.This technology can be suitable for gene substitution.
The conventional homologous recombination method that is different from many use selective markers, used the method for series connection superinduce HR to introduce and to remove subsequently the box that comprises anti-selective marker by single transformation event.For example, carry the box of the sequence of anti-selective marker and generation series connection repetition and can in yeast host, by conversion, introduce the donor gene group, then the spontaneous homologous recombination between the homology zone in box and genome is selected.
Can be as above-mentioned, for example by the situation that URA3 in the situation that do not exist Histidine to grow, initial mark is introduced and is selected.Transitional cell enters anti-selection substratum and (for example, 5-FOA), by the homologous recombination between tandem repetitive sequence, spontaneous mark " is given prominence to " and selected subsequently.Such method can be applicable to disappearance, point mutation and gene substitution by changing with the box part of target nucleic acid homology.
This paper shows that introducing series connection in conjunction with conventional recombination method repeats, and for " giving prominence to ", in yeast, modifying some donor nucleic acid, is restricted.See following embodiment 4B (ii).This restriction is the high background due to the spontaneous loss of negative (instead) selective marker.The method provided has been improved efficiency and has been reduced unwanted background sudden change (for example, spontaneous deletion) and be used in yeast and modified bacterial genomes in host cell.
Iv. series connection repetition-endonuclease cutting (TREC)
Near series connection repetition target region and enzyme cut the two and all can be used for helping to remove selective marker.A kind of such embodiment, be called " cutting of series connection repetitive nucleic acid restriction endonuclease " (TREC) method, be combined with the conventional homologous recombination replaced of anti-selective marker, expression by endonuclease target region or near induce the introducing double-strand break, and introduce tandem repetitive sequence at the flank of the nucleotide sequence that comprises mark.The method can be used for accurately efficient bacterium donor gene group and the large nucleic acids that is cloned into yeast host cell of modifying.
Utilize the TREC method, the efficiency in conjunction with having strengthened widely the target-specific restructuring of the tandem repetitive sequence of flank and double-strand break nearby or contiguous also allows genetically modified bacteria genome in yeast host.Wherein use the method successfully the seamless example that has lacked CD139 locus in the mycoplasma genitalium genome of growing in yeast in embodiment 4C, describe below.The method is illustrated in Figure 10 C.
Method described herein can be used for introducing any modification, such as point mutation (for example, nucleic acid and codon are replaced, comprise conservative and non-conservative replacement), disappearance, insert and to the donor nucleic acid target region in host cell, such as other modifications of the bacterial genomes in yeast host cell.
V. the generation of target box (targeting cassette) and box
Provide for design and generated for example, method for the nucleic acid (, target box) of modifying method.Construct and other nucleic acid for the method also are provided.Usually, comprise with the part of donor target region homology and (partly compare with homology for the target box of introducing selective marker, it optionally comprises one or more sudden changes, disappearance, insertion, replacement or other modifications) and selective marker, anti-selective marker typically, such as URA3.Usually, this box comprise with the 5 ' homeologous part of target region and with the 3 ' homeologous part of target region.
In some embodiments, generate the second target construct, to use the replacement nucleic acid selective marker had with the target nucleic acid homology.The homologue that this second target construct comprises target region or its part is also partly compared with homology at target region, optionally comprises one or more sudden changes, disappearance, insertion, replacement or other modifications.
In some embodiments, for near target region, in target region or contiguous target region introduce double-strand break, the target box also comprises codase such as cut the gene of nucleic acid such as the endonuclease of dsDNA in particular sequence, and also comprise the nucleotide sequence of being identified by enzyme, its usually the box end or near.Usually, the gene of coding nucleic acid restriction endonuclease is at inducible promoter, under the control such as the GAL1 promotor, so that can be by under certain environmental conditions, and such as in the situation that exist semi-lactosi as sole carbon source, growth host cell, inducible gene expression.
In some embodiments, in order to generate tandem repetitive sequence, the target box comprises the other part with the target nucleic acid homology, and it is along the upstream of the target region of target nucleic acid length or downstream, once, in order to integrate box by homologous recombination, tandem repetitive sequence will exist in target nucleic acid.
When using cutting and tandem repetitive sequence, this box comprises the first homology part, itself and the portion homologous of the target nucleic acid in the upstream of target region along target nucleic acid length or downstream (with the generation repetition of connecting); The second and the 3rd homology part, its respectively with 3 ' and the 5 ' portion homologous (for by homologous recombination, inserting box) of target region; The nucleic acid of codase (for example, endonuclease) under inducible promoter is controlled; The nucleotide sequence of endonuclease identification; And selective marker, typically anti-selective marker.Usually, the second and the 3rd homology part (with target region 3 ' and 5 ' portion homologous) is at the flank that comprises the first homology partial sequence (it generates the repetition of connecting).On the one hand, the second and the 3rd homology part is also for example, at the flank of the nucleotide sequence that comprises codase (, endonuclease).They are typically also at the flank of the sequence that comprises selective marker.
On the one hand, the nucleotide sequence of being identified by enzyme is positioned at contiguous the second or the 3rd homology part relative the first homology part (it generates the Tandem repeat territory) the relative end at construct.
On the one hand, with homology in target nucleic acid, partly compare, one of the second or the 3rd homology part (for integrating box) or both comprise one or more coding mutations, insertion or disappearance.
The exemplary target box used in the TREC of embodiment below is illustrated in Figure 10 C.This exemplary constructions body comprises the I-SceI recognition site, under the GAL-I promotor is controlled coding I-SceI endonuclease the anti-selective marker of nucleic acid, URA3 and with the part (marking " repetition ") of part (mark " repetitions ", also describe in the target nucleic acid) homology of the target nucleic acid sequence of target region upstream.This box also comprise with the part of 5 ' the 50bp for the I-SceI site of target region homology and with the target region homology be " repetitions " homology 50bp part of 3 ' partly.The target box that comprises recognition site, endonuclease-encoding gene and inducible promoter and selective marker partly is called " CORE " box.Described in embodiment 4C below, this box, at host cell, by 450 base pair parts of missing gene group target region, is modified mycoplasma genitalium donor gene group, and described donor gene group has been used the method provided to be transferred in yeast host.Similarly construct is for being used the method provided at the II type restriction enzyme allele of yeast host cell disappearance in mycoplasma mycoides LC genome.
The modification of these target boxes, such as described at Figure 10 A-D and this paper elsewhere described those, the method provided also is provided.For example, the modification of this box can be used for sudden change, replacement, insertion and other modifications such as the Nucleotide of modifying is introduced target nucleic acid.The remodeling of this box provided and method will be apparent for the technician.
Can use that any much nucleic acid of knowing is synthetic, amplification, connection and assembly method, such as those and commercial available method described herein generate this box.In one embodiment, form the nucleic acid fragment generation box of this box part by amplification and/or assembling.The method that can know by use, for example, such as chemosynthesis or comprise from plasmid, genomic dna or other nucleic acid amplification of expecting nucleotide sequence and (, PCR) generate this fragment.
On the one hand, use to merge PCR, use the recombinant PCR technology, as at Shevchuk, N.A. etc., Nucleic Acids Res, describe in 32, el9 (2004), and the assembling fragment is to form box.
Utilize the method, use chimeric fusion primer, the two kinds of different fragments to be connected that increase, then connecting without primer (primer-less) polymeric enzyme reaction (for example,, without primer PCR) is middle.Each chimeric primers comprise with the part of the first fragment homology to be connected and with the part of the second fragment homology to be connected.Therefore, use two fragments of primer amplification to generate overlapping homology zone between amplified production, such as the 40bp homologue at the product end.
These products for example then are used in, in many (, 10,11,12,13 or more) PCR circulation that does not have primer and use, with low temperature thermal oxidation, such as at 56 ℃ or about 56 ℃, with by overlapping extension connection product.The many products that connect in this mode then can connect in fusion PCR step subsequently.Usually, fusion product increases in other PCR reaction again, such as use is included in the primer of the end of expectation box other sequence to be added.
Other assembly methods are known, and can be used for generating box.For example, use conventional synthetic method as described to prepare this box, or can buy from commercial supplier.Can use other assembly methods, such as passing through a step isothermal dna assembly method of 5 ' exonuclease, archaeal dna polymerase and DNA ligase teamwork, it is described in Gibson etc., NatureMethod 6,343-345 (2009) and in the Application No. 12/371,543 of submitting on February 19th, 2009.Utilize the method, at first DNA fragmentation caves in (recess) by 5 ' exonuclease, produces the strand overhang, and it is annealing specifically then, then uses polysaccharase and ligase enzyme fill up the gap covalently bound.Other assembly methods are described in U.S. Patent Application Publication No.: in US2007/0037197A1 and US2007/0037196A1.
Vi. transform and modify analysis
In order modifying, box to be transformed into the host cell of donor gene group such as those donor gene groups of the generation of the method according to providing is provided.Method for transformation is known.In one embodiment, according to disclosed method (Gietz, D. etc., Nucleic Acids Res, 20,1425 (1992)), with 2-3 μ gPCR product and 25 μ g carrier DNA (Salmon testis DNA, Sigma, St.Louis, MO), use the lithium acetate integration transformation box to be introduced to the yeast host cell that comprises the donor gene group.
In order to select box to be integrated into the cell of target nucleic acid, cell is grown and is selected independent URA in lacking the substratum of uridylic +Transformant is also optionally passed through pcr analysis, and it uses and inserts the diagnostic primers of the target donor nuclei acid moieties specific binding of regional flank at box.By using existence and the size of this primer assessment amplicon, determine whether box correctly inserts, and whether described primer inserts the amplicon that produces different sizes according to box.An example is described in embodiment 4C (ii) below.Use the cell that comprises correct insertion in the later several rounds homologous recombination.
When carrying out enzyme induction and express to produce ds (two strands) fracture, the cell that comprises anti-selective marker is then grown inducing the condition of expressing enzyme from inducible promoter, such as growing in comprising the substratum of semi-lactosi.In one embodiment, cell is grown comprising on the SG of semi-lactosi as sole carbon source (synthetic semi-lactosi)-His substratum, for example, 4 hours or 24 hours, to induce the expression of the enzyme of introducing double-strand break.Can carry out growing comprising on the substratum of glucose, in contrast.
In some embodiments, the second nucleic acid is transformed into to cell, this second nucleic acid comprises the sequence with the target nucleic acid homology that will replace selective marker.In some cases, with target nucleic acid, compare, this second nucleic acid comprises one or more sudden changes, disappearance, insertion or replacement.See embodiment 4A and 4B.In other cases, after insertion vector, the series connection generated in target nucleic acid repeats, and the second homologous recombination event spontaneously occurs.Utilize the TREC method, the combination of these methods is for removing selective marker.
By growing being beneficial under the condition of loss, select the loss of anti-selective marker.In some aspects, as URA3 when marking, before this selection (for example, second take turns conversion after), cell is grown existing in the uridylic situation, under 30 ℃, spend the night, in thering is the yeast cell of losing the URA3 gene, to exhaust residual Orotidine-5 '-'-phosphate decarboxylase (by the URA3 genes encoding).Then being conducive under the condition of losing, under at 5-FOA, having situation, such as the cell of the anti-selective marker of selecting to have loss on the HIS flat board comprising 5-FOA, to select the loss of URA3 gene.Can be used identical or different diagnostic primerses, at the pcr analysis of insertion point flank, with the disappearance of checking box.
Can carry out multiplex PCR to analyze donor nucleic acid such as the genomic integrity that the modifying method modification provided is provided.For example, can be as at D.G.Gibson etc., PNAS USA, implement multiplex PCR (MPCR) described in 105:20404-9 (2008).
For PCR and MPCR analysis, can use separation method described herein to carry out separating all DNA from host cell according to the type of host cell.In one embodiment, as described, carry out isolation of genomic DNA from yeast host cell in embodiment 3.Can design and have different sizes, along donor gene group length, around ring-type bacterial genomes in yeast, a plurality of parts place has the MPCR primer sets of homology, in order to can verify the existence of each amplicon.See, such as D.G.Gibson etc., PNAS USA, 105:20404-9 (2008)).Can use the method for knowing, comprise commercial available test kit, such as the Qiagen multiple PCR reagent kit carries out multiplex PCR.Exemplary reaction is described in following embodiment 4A.The genome that the existence indication of each amplicon is modified is completely, and typically carries out, and to guarantee that spontaneous unwanted recombination event does not occur, generates unwanted modification.
According to donor, host and recipient cell type, can use other modifying method in conjunction with the method provided.For example, can use the Cre-LoxP system of knowing.The Cre-LoxP system is known effective locus specificity recombination method, and it has been successfully used to remove selective marker and large genomic DNA fragment in many different biologies.As described for additive method, for example, by two-wheeled PCR reaction, can produce the Cre-LoxP mutagenesis construct with mutant LoxP gene.As at Araki, K. etc., Nucleic Acids Res, described in 25,868-872 (1997), the LoxP sudden change prevents reverse recombination event.Example is described in following embodiment 4D.In one embodiment, modifying method is compared with the modification by the Cre-LoxP system, equally effective, basically equally effective or more effective.
F. shift donor gene group and the nucleic acid modified and enter recipient cell
This paper provides for transfer donator nucleic acid, comprises the method that supplies Autosome and/or donor gene group to enter host cell or recipient cell.Donor nucleic acid can be transferred to recipient cell from host cell.Donor nucleic acid is included in those that modify in host cell.In another embodiment, the donor gene group directly is transferred to recipient cell from donorcells, for example by shifting the natural gene group, enters recipient cell.The donor gene group that transfer method can be used for effectively will breeding in host cell and modify is transferred back to gene product therein can be from the environment of genomic expression.The cell that recipient cell and donorcells or biophase ratio can be mutually of the same race or be closely related and plant.
For cloning the small nucleic acids fragment, such as gene fragment, enter host cell and be known by the method that they are transferred back to initial or the cell that is closely related, but be generally limited to operate small nucleic acids, for example modify the mononucleotide fragment, it is followed separated and inserts the genome return to initial donorcells.As Lartigue etc., Science 317,632 (2007) is described, and the full genome of mycoplasma has successfully directly been transferred to the not of the same race acceptor mycoplasma cell be closely related from donor mycoplasma cell, once shift the successful expression gene product.
But, (for example shift large nucleic acids with regard to it from host cell, genome and karyomit(e)) to the ability of recipient cell, available transfer method is restricted, the host cell that described recipient cell has been bred therein with genome is compared and is not closely related, such as, be the cell of different biological branches.For example, available method is restricted for transfer to former nuclear receptor from eucaryon host.
For example, the protokaryon donor gene group that makes to breed in eucaryon host is transferred back to that former nuclear receptor can be subject to that nucleic acid reclaims, methylates, uncompatibility and toxicity problem limit.Need such method, wherein from host cell, reclaim q.s donor nucleic acid purifying, complete, to generate q.s, such as the recipient cell that comprises transfer donator nucleic acid of detectable amount.
Once shift the donor nucleic acid bred in host cell, in recipient cell, exist (with perhaps also existing in donorcells), but in host cell, non-existent restriction modification system can cause uncompatibility.For example, because the yeast host cell of yeast saccharomyces cerevisiae contains the restriction modification system existed in some bacterial cells, the bacterial genomes of separating after growth in yeast host can suffer restriction modification system (one or more) impact (Holt etc. of bacterial receptor cell, Bioessays 29,580 (2007).Therefore, by yeast cell, modify and the bacterial genomes of propagation shift enter (such as donorcells and other bacterial receptor cells) in the cell of expressing therein the donor gene product have transfer genome can with the inconsistent risk of recipient cell.
In addition, but these yeast hosts that do not comprise restriction modification system are the expressible dna methyltransgerase still, and it can modify donor nucleic acid (such as bacterial genomes), once shift, enters recipient cell such as bacterium, suppress their activation (for example, gene product expression).
In addition, in host cell propagation and modify after, the structure of the donor gene group of separation and conformation can be different from genomic conformation and the structure of breeding in cell more being closely related with the donor biology.These differences can adversely affect donor nucleic acid is transferred back to recipient cell.Transfer method described herein comprises the aspect that overcomes these restrictions, for successfully modifying and/or the donor gene group of breeding is transferred to recipient cells different in heredity at host cell, such as from eucaryon host to former nuclear receptor.Among these aspects is to methylate in vitro, process the degraded host cell proteins, also shift and enter the recipient cell that lacks restriction modification system with enzyme, such as these systems by the mutant receptors cell.The exemplary research that provided transfer method success is provided is described in detail below in embodiment 3 and embodiment 5.
Fig. 8 schematic illustrations three aspects of the transfer method that provides.In the first method (meaning with " 1 " mark arrow), the middle separation donor dna of the agarose inserted block melted---such as processing with β-gelase---, and directly be transferred to recipient cell.Shift between similar cell when nucleic acid and while not considering the uncompatibility problem, typically use this first method.In the second method (" 2 " mean), recipient cell is modified the restriction enzyme that suddenlys change, then as in the first method transfer, moved donor nucleic acid.In third method (" 3 " mean), the donor nucleic acid experience in the agarose inserted block methylates and the deproteinization reaction, then melts and shifts, and exempts from the impact of acceptor R-M system and conformational change with protection donor nucleic acid.On the other hand, methylated and be there is no deproteinization.
Figure 16 schematic illustrations the other aspect of the transfer method that provides.Bacterial genomes can move into yeast, transformation and shift and install back bacterium by genome.Yeast vector can insert bacterial genomes by conversion; This bacterial genomes is cloned into yeast.After the clone, use all technology of yeast genetic method to manufacture one or more insertions, disappearance, rearrangement or its any combination in bacterial genomes.The genome of this transformation can be followed separated and be transferred to recipient cell to generate the transformation bacterium.Before transfer, in some cases, the donor bacterium DNA that methylates can be necessary, to protect it, avoids the impact of the restriction system (one or more) of recipient cell.This circulation can start repetition (dotted arrow) from the genome of new transformation with iterative manner.
I. separate donor nucleic acid from host cell or donorcells
In first step, separate donor nucleic acid (for example, donor gene group) from host cell or donorcells.For knowing from the method for cell isolating nucleic acid, comprise for separating of genomic dna, comprise complete genomic method and for separating of the method for organelle gene group.Any such method, comprise described herein those, can be used for separating donor nucleic acid.Should be appreciated that the selective dependency of method is in nucleic acid type to be separated and its cell type from wherein separating.
Usually, described in embodiment below, carry out large nucleic acids such as genomic separation in the agarose inserted block.
Several aspects of the transfer method of describing provide effectively and the high quality transfer nucleic acid of high yield.On the one hand, the cell that comprises donor nucleic acid in the situation that have paraxin or similar substance is grown, then separates donor nucleic acid.Paraxin obtains donor gene group and karyomit(e) compact and that fully copy for the nucleic acid samples (such as the agarose inserted block) separating.Make that ongoing to copy wheel subsynchronous, suppress (Drakulic and Errera, the Biochim Biophys Acta 31,459 (1959) of copying of next one; Skarstad etc., EMBO J 5,1711 (1986); Bernander etc., J Bacteriol 111,1670 (1995); Skarstad etc., at Flow cytometry applications in cell culture A.N.E.Mohamed Al-Rubeai, Ed. (CRC Press, New York, 1996) pp.241-255) and compact nucleoid (Murphy and Zimmerman, J Struct Biol 133,75 (2001); Seto and Miyata, J Bacteriol 181,6073 (1999)) be known.In the mycoplasma culture, the existence of paraxin can help to obtain genome compact and that fully copy in the agarose inserted block.
A. from host cell, separate
In the situation of separating from host cell at donor nucleic acid, donor nucleic acid can be used the scheme compatible with host cell to separate in the agarose inserted block.On the one hand, when host cell is yeast, can for example use CHEF mammalian genes group DNA inserted block test kit (Bio-Rad), the specification sheets extracted for cerevisiae dna of recommending according to manufacturers, used optional improvement, prepares the agarose inserted block.In a kind of non-limiting example, the culture of the yeast host cell that comprises bacterium donor nucleic acid is grown until OD under 30 ℃, in selective medium 600Reach about 1.5.In one embodiment, every mL inserted block is used 6 * 10 9Individual yeast cell (replaces 6 * 10 of manufacturers's recommendation 8Individual cell) to increase the amount of the obtainable donor nucleic acid of each inserted block.The cell walls that is embedded in the yeast host in the agarose inserted block is digested.Can use lyticase (Biorad), recommend according to CHEF test kit manufacturers, or use 100T (β-1,3-dextran kelp pentasaccharides lyase (β-1,3-glucan laminaripentaohydrolase); USB, Cleveland, OH) carry out cell walls digestion.In one embodiment, Zymolase TMEnzyme adds the inside or the outside of inserted block to the concentration of 5mg/mL.Mixture is placed 2 hours under 37 ℃.
In one embodiment, 1 * TE damping fluid (20mM Tris-HCl, pH 8; 50mM EDTA) after middle rinsing, by 5ml Proteinase K reaction buffer [the 100mM EDTA with being supplemented with every ml inserted block 200 μ l Proteinase Ks; 0.2% Sodium desoxycholate; 1% sodium lauroyl sareosine; PH8.0] at 50 ℃ of lower incubations twice 24 hours, the yeast cell of cracking embedding digesting protein.The agarose inserted block at room temperature uses 40ml 1 * TE damping fluid to stir rinsing 4 times, each 1 hour.Sample then is kept in identical damping fluid under 4 ℃.In some cases, be desirably in step subsequently the nucleic acid that digestion separates, such as for removing host DNA or linearizing donor gene group.Under these circumstances, add the phenylmethylsulfonyl fluoride (PMSF) of 1mM during rinsing for the second time.
When donor nucleic acid is the organelle gene group, for isolated cell device genome, revise separation scheme, the organelle gene component is from method as discussed in this article.
B. remove host's nucleic acid
In order to separate donor nucleic acid from host cell, may expect to remove host's nucleic acid.In one embodiment, for separation of bacterial donor gene group from yeast host cell, pastoris genomic dna is also separated together with the bacterial nucleic acid extracted from host cell.On the one hand, separate and comprise " cleaning " step, wherein remove pollutent host nucleic acid, such as using recognition of host nucleic acid but the restriction enzyme of nonrecognition donor nucleic acid.In one embodiment, in order to remove the pollutent pastoris genomic dna, the restriction enzyme mixture process of specificity digesting yeast genomic dna for inserted block.
In one embodiment, by for example, by inserted block and specificity cutting host genome DNA but (retain restriction enzyme that donor dna is complete, 50 AsiSI of unit, RsrII and FseI enzyme (New England Biolabs in 500 μ L reaction volumes, Ipswich, MA)) incubation that spends the night under 37 ℃, removed endogenous host DNA inserted block.Inserted block then at room temperature use 1ml 1 * TE damping fluid rinsing 1 hour application of sample to 1%TAE sepharose (120 minutes, 120 volts) to remove the host DNA fragment of digestion from inserted block.
In another kind of embodiment, at host genome DNA, be linear and the donor gene group is in the situation of ring-type, remove host genome DNA by the pulsed field agarose gel electrophoresis, whereby host genome DNA be retained in well and donor gene group DNA by electrophoresis outside this hole.(Lartigue etc., Science 317,632 (2007)).In a kind of such embodiment, the yeast inserted block is used the CHEF DR IIII from Bio-Rad, with clamper uniform electric field (Chu etc., Science 234,1582 (1986)), at 1% LMP gel, carries out electrophoresis in 1 * TAE damping fluid.Usually, under 3.5V/cm, the burst length, from 60 slow rise (ramp) by 120 seconds, continues 24 hours.After electrophoresis, remove inserted block and be stored in 1 * TE damping fluid under 4 ℃ from hole.
After by either method, separating host DNA, can from hole, remove the agarose inserted block, for further processing.In one embodiment, the inserted block of removing in 1mL 0.1 * TE damping fluid rinsing twice 1 hour and in 1mL is supplemented with 1 * NEB damping fluid 2 (New England Biolabs, Ipswich, MA) of BSA (100 μ g/mL) balance 1 hour.In order to make the DNA linearizing of donor gene group so that its electrophoresis on sepharose, can be by inserted block incubation together with restriction enzyme.In one embodiment, inserted block and 50 PspXI of unit restriction enzymes are incubated overnight under 37 ℃.After incubation, with 1mL 1 * TE damping fluid at room temperature rinsing inserted block 1 hour application of sample to pulsed field gel.
In another kind of embodiment, before transforming, do not remove host DNA.
C. from donorcells, separate
In another embodiment, for example, at donor nucleic acid (, the donor gene group), directly from donorcells, be transferred to the situation of recipient cell, used the separation method compatible with donorcells.In one embodiment, there is in order to separate the donor bacterium genome from donorcells, to use the agarose inserted block that improved CHEF mammalian genes group DNA inserted block test kit (Bio-Rad) preparation comprises genomic dna.
In one embodiment, cell (for example, the mycoplasma mycoides LC cell that comprises the donor gene group or the yeast cell that comprises the donor gene group) is grown in suitable medium, until the OD of expectation, and, then by itself and the paraxin incubation of 100 μ g/ μ l 90 minutes, then gather in the crops.
For separate the exemplary arrangement of complete complete genome DNA from the mycoplasma donorcells, as Lartigue etc., Science 317,632 (2007) is described to carry out, and is optionally improved, such as before separation, cell culture being modified.A kind of such example is described in embodiment 2B (ii).
D. separate the quantification of donor nucleic acid
Before shifting, can quantize or assess the amount of the donor nucleic acid separated.In one embodiment, the donor nucleic acid separated from host cell in sepharose electrophoresis and with the donor nucleic acid that separates from the known quantity donorcells relatively.An example is described in embodiment 2B.In another embodiment, such as the amount of the donor nucleic acid separated by the quantifying of UV spectrophotometric.A kind of such example is described in embodiment 2B (iv).
Ii. process donor gene group and/or the recipient cell separated
The method provided comprises for overcoming the step of uncompatibility obstacle between host, donor and recipient cell/nucleic acid.Described in this article this obstacle, this obstacle can limit large nucleic acids such as the donor gene group is transferred to from host cell the recipient cell that the donor gene product can be expressed therein.For example, when host cell and donor and receptor biological are not (, from different biological branches) be closely related, especially this situation.Be transferred to former nuclear receptor for the Prokaryotic genome that will breed in eucaryon host, this obstacle is related (relevant).
Many factors comprise that uncompatibility and toxicity can cause this obstacle.For example, once shift the donor nucleic acid of having bred in host cell, the restriction that have (perhaps also existing) in recipient cell, still in host cell, does not have in donorcells-modification (R-M) system can cause uncompatibility.Restriction modification system is known, and usually by bacterium living beings, is used to protect biology to avoid the impact of foreign DNA.Restriction modification system generally comprises protein and for example, enzyme so protection those sequences in biology self nucleic acid for modifying (, methylating) of the particular sequence for identifying and cut foreign DNA.Restriction modification system comprises I type, II type and III type system.I type system generally comprises identification (specificity) respectively, cutting (restricted) and modifies the complex body of three kinds of protein of (modified) nucleotide sequence.Therefore, this complex body methylates and cutting DNA.II type system generally comprises two modifying enzymes that separate and restriction enzyme, methylates respectively and the cutting DNA sequence.Restriction enzyme and modifying enzyme that III type system comprises the complex body that forms heterodimer, for modifying and cutting.Can also methylate their DNA of modifying enzyme.
In addition, after being transferred to recipient cell, the dnmt rna that host cell (comprising those that do not comprise restriction modification system) is expressed can be modified donor nucleic acid and suppress their activation (for example, gene product expression).In host cell, after propagation and modification, the structure of donor nucleic acid and the variation of conformation can adversely affect donor nucleic acid and be transferred back to recipient cell.
The transfer method provided comprises for having overcome the step of these restrictions, with the donor gene group that successfully will modify in host cell and/or breed, is transferred to recipient cells different in heredity, such as from eucaryon host to former nuclear receptor.Step comprises donor nucleic acid and (2) modified receptor cell that (1) processing separates.
A. in vitro tests is to estimate the uncompatibility of restriction modification system
Can utilize in vitro tests determine between host cell, donor nucleic acid and recipient cell whether for example due to different biologies between the discordance of restriction modification system have the uncompatibility problem.The restriction modification system of donor gene group or receptor expressing cell can have slacken successfully shift and in host cell, activated donor is genomic may.
Such as the possible restriction of the bacterial genomes of breeding-modification problem, with regard to the restriction modification system aspect, estimate donor, host and/or recipient cell in order to check the donor gene group of breeding in the host cell of dissimilar biology in yeast.In donor and/or acceptor, exist (with the recognition site specificity of system) of restriction modification system can be used known method to identify from donor gene group sequence.Also referring to REBASE---the restriction enzyme database, can be in Website: in rebase.neb.com/rebase/rebase.html, obtain.
For further confirming the existence of R-M system, the existence that can test in vitro modifying enzyme.For this process, the methylation state of the recognition site of prediction can be used the restriction enzyme in commercial available identification prediction site to survey.For example, can use the restriction enzyme isoschizomer of the restriction enzyme system of commercial available corresponding prediction in digestion reaction, whether methylate at suitable restriction site to determine donor and acceptor gene group.Can protectedly avoid the enzyme cutting in this site of commercial available identification at the methylated genomic dna in prediction site.If protected avoiding of acceptor gene group DNA cuts, the existence of the modifying enzyme of susceptible of proof R-M system.Also can carry out this process at the genomic dna separated from donorcells, in order to estimate the protected systematic influence of avoiding of donor gene group possibility.An example is described in following embodiment 2D.
In addition, the cell-free extract prepared from acceptor and donorcells can be used for determining whether have the restriction enzyme of prediction and activity is arranged in cell.Method for the preparation of cell-free extract is known, and can with together with provided method, use according to cell type.In a kind of non-limiting example, as (b) middle description of following embodiment 2D (ii), preparation is without the mycoplasma cell extract.Comprise the DNA that predicts restriction site and cell-free extract in the restriction digest incubation to determine existing at the extract of the enzyme of this recognition sequence cutting DNA.The sample of digestion can be in sepharose electrophoresis to determine cutting.If expectation, can compare in test at specific site and methylated or there is no methylated DNA.By adding EDTA, such as 10mM EDTA, cell-free extract also can be used as the methyl transferase activity source, to suppress nuclease.Alternatively, before digestion trial, the restructuring methyltransgerase, such as intestinal bacteria dam methyltransgerase (New England Biolabs, Ipswich, MA) can be used for methylate DNA.Methyltransgerase also can be purified.The example of this digestion trial be described in following embodiment 2D (ii) (b) in.
B. donor nucleic acid methylates
From the donorcells after separating and before being transferred to recipient cell, donor nucleic acid can be methylated in vitro.The donor gene group that methylates, such as in host cell, bred those, can protect them to avoid the restriction modification system of host cell and/or by those impact of donor gene group coding.On the one hand, provide as this method, its protection donor nucleic acid of having bred in host cell is avoided the R-M system of acceptor and by those impact of donor coding.Other aspects, provide protection donor gene group to avoid the method for the R-M systematic influence of one of these biologies.For example, in many cases, by the enzyme donor gene group that methylates, be possible, it protects it to avoid the impact of acceptor R-M system.Before the lethal concentration of corresponding donor restriction enzyme reaches, when donor nucleic acid is methylated by the donor methyltransgerase, be exactly this situation.
As following described in the embodiment 2; when being transferred to the mycoplasma capri recipient cell; there is no need to protect mycoplasma mycoides LC donor gene group DNA to avoid its restriction system impact, before the lethal level of this hint restriction enzyme activity reaches, the donor gene group is methylated.This is not strange, because most of endonuclease and methyl transferase gene are to can be cloned into intestinal bacteria simultaneously.See Holt etc., Bioessays 29,580 (2007).Should be appreciated that the restriction modification system aspect with regard to them, estimate donorcells, host cell and recipient cell whether protected before shifting to estimate the donor gene group.
When from yeast, shifting other bacterial genomes; the donor gene group that methylates in vitro may be necessary with the impact of protecting it and avoiding its restriction enzyme; such as by using cell-free extract or methylating in vitro or, by make the restriction endonuclease gene inactivation the donor gene group, described as following from the methyltransgerase of donor purifying.As described below, externally methylate and limit digestion to can be used for determining in concrete transfer research to need which kind of methylation reaction.
Usually, use methyltransgerase to be methylated, described methyltransgerase is same or similar with those methyltransgerases of the R-M system of protecting from expectation.Can use the methylase for example, separated from cell extract (, recipient cell extract) or the methylase with purifying produced of recombinating to be methylated.
On the one hand, the methylase heterogenous expression of the R-M system of acceptor and/or donorcells use recombinant chou method purifying.For all methyltransgerases, by the encoding sequence of R-M Forecasting recognition, can with the system in expectation, such as yeast or bacterial cell, be expressed by codon optimized.Can use any many synthetic and/or assembly methods of knowing, such as described herein those, build the fragment that comprises encoding sequence.Non-limitative example be described in embodiment 2E (i) (a) in, wherein use a step isothermal dna assembly method to generate methyltransgerase-coding nucleic acid.After structure, fragment is cloned into to expression vector, for the cells of selecting.
Carrier can be used for transformant with the expressing gene product.Exemplary expression system comprises just (codon plus) cell (Stratagene, La Jolla, CA) of BL21 (DE3) codon.Can in cell, induce the Methyl transporters expression of enzymes, such as by incubation together with IPTG.Can use the method for knowing, purifying methyltransgerase from cell.In one embodiment, clarification cell lysate, post-purifying, and the fragment that comprises methyltransgerase is dialysed to use in methylation reaction subsequently, such as 50mM HEPES-NaOH pH 7.2,50mM NaCl to enzyme buffer liquid, 0.1mM EDTA, 10% glycerine).If necessary, can follow concentrating sample.Exemplary expression and purification schemes embodiment 2E (i) are below described in detail in (b).
On the other hand, the crude extract from the cell with R-M system (such as recipient cell or donorcells) is used for the donor nucleic acid that methylates and separate from host cell.If all R-M systems of uncertain acceptor or host cell are determined, this aspect can be favourable.For example, if the R-M system of recipient cell is unknown, uses so from the crude extract of recipient cell and will guarantee to exist all relevant methyltransgerases in methylation reaction.Can use any method for the preparation of applicable cell extract of knowing.An example is described in following embodiment 2E (ii), for the cell crude extract that comprises methyltransgerase from the preparation of mycoplasma recipient cell.Nuclease in cell extract can be suppressed, such as passing through to add 10mM EDTA, so that they use in methylation reaction.
The donor nucleic acid that the methyltransgerase of purifying or the cell crude extract that comprises methyltransgerase can be used for methylating and separate from host cell.In one embodiment, the agarose inserted block that comprises donor nucleic acid can for example, at the damping fluid that methylates (, 100mM Tris-HCL pH 7.5; 10mM EDTA; 3 μ M DTT; 200 μ M S-adenosylmethionines (SAM)) rinsing and balance in.Then, inserted block can be in the methylation reaction of the methyltransgerase that comprises methylate damping fluid and cell crude extract or purifying incubation.Do not have the parallel reactor of SAM to can be used as contrast.On the one hand, can in the situation that has dam methyltransgerase (New England Biolabs, Ipswich, MA), carry out methylation reaction.After methylating, each yeast inserted block can be in the 1ml Proteinase K reaction buffer that is supplemented with 40 μ l Proteinase Ks, 50 ℃ of lower incubations 4 hours.Inserted block can be followed rinsing 4 times 45 minutes, uses 1ml 1 * TE damping fluid at every turn, and at room temperature rinsing 2 times 30 minutes, at every turn in 0.1 * TE damping fluid, and gentle the stirring.After removing final rinsing damping fluid, can melt filler.An example is described in embodiment 3A (iii).Before transfer research, can test in vitro methylation reaction and avoid the effect of acceptor R-M systematic influence for protection donor nucleic acid.According to R-M system evaluation result, can make adjustment.Can, by donor gene group DNA or the plasmid that comprises donor nucleic acid are implemented to methylation reaction, carry out this process.After methylation reaction, can use the restriction enzyme of recipient cell to be limited digestion reaction, to guarantee by the protection donor nucleic acid that methylates.An example is described in embodiment 2E.
C. deproteinization
The conformation of donor dna can be changed with the crude extract incubation, once shift it, uncompatibility can be caused again.Can be by visual in the situation that exist or do not exist the donor gene group DNA of cell extract incubation to estimate this conformational change, such as described in embodiment 2B (iv).Therefore, in some embodiments, after methylating and before shifting, donor nucleic acid can carry out the Deproteinization step, to remove deproteinize in crude extract.On the one hand, can use proteolytic enzyme such as Proteinase K is removed protein.In exemplary process, carry out the agarose inserted block of methylation reaction can be further at Proteinase K reaction buffer and Proteinase K 50 ℃ of lower incubations 4 hours.Before continuing to melt inserted block and shifting, but the rinsing inserted block.See embodiment 2B (iv) and 3A (iii).
D. genetic modification recipient cell R-M system
In some cases, before transfer donator nucleic acid or genome, the restriction modification system of recipient cell may need by inactivation; Can in vitro or carry out the modification of R-M system in body.Replace methylating in vitro, restriction modification system can be at least from recipient cell with remove or inactivation from donor gene group or nucleic acid possibly.
One or more restriction enzymes (one or more) of recipient cell R-M system can be by the gene inactivation of sudden change codase.This process usually can be used as the alternative of the donor nucleic acid that methylated in vitro before shifting.
But the restriction modification system inactivation of donor gene group, may be unpractiaca in some cases.The restriction enzyme of for example, expressing at once after shifting by the donor gene group coding can help by resident's genome of degraded recipient cell to promote to shift.Therefore, in this situation, be to remove the donor restriction modification system not expect, should use and methylate.Should be appreciated that before shifting each that can estimate with regard to this point donor, host and recipient cell, to identify the best system for inactivation R-M system.
Any mutation process can be used for inactivation R-M system.An example is described in following embodiment 2F, and wherein, before transfer donator nucleic acid, in the mycoplasma recipient cell, the gene of coding Single restriction enzyme is by inactivation.The tetracycline resistance marker of the cell that in this embodiment, gene allows selection to comprise mutator gene by use interrupts being suddenlyd change.This paper considers other method for deactivating and comprises the resistance marker of many cells that also can be used for selecting comprising mutator gene; This paper describes that such mark and they are also known in this area.The cell extract prepared from this mutant recipient cell can be with comparing, as described herein in methylation reaction.
Alternatively, the donor gene group can be methylated in vivo, for example, and when also in host cell.Enter the donor of host's carrier or the methylase of acceptor by cloning by expression, can carry out methylating in body in host cell.This respect may more not expected, because it may cause the variation of not expecting in the donor gene group.For example, express the bacterium methylase and shown the homologous recombination that has increased yeast in yeast, it can cause the donor bacterium genome that is arranged in yeast artificial chromosome (YAC) or yeast centromeric plasmid (YCp) to change.This result can occur, and this is because yeast host cell does not have the integrity of selective pressure maintenance bacterial genomes, except the insertion yeast vector sequence area of described bacterial genomes.Should be appreciated that and can estimate the R-M system that externally methylates, methylates in body or whether can be used for the inactivation recipient cell by inserting resistance marker inactivation R-M system for example.
Iii. be transferred to recipient cell
After separating and processing, donor nucleic acid can further be used described herein or methods known in the art to be transferred to recipient cell.An exemplary transfer scheme is described in following embodiment 3.For shifting the mycoplasma genome from donor to a method of mycoplasma acceptor by Lartigue etc., Science 317,632 (2007) describes.Such method is used in host separately modifies from difficult cell or biological genome or nucleic acid to give the donor gene group specific character.The genome of modifying can then be transferred back identical donorcells or enter donorcells, thereby gives the phenotype of recipient cell modifying factor group.
Usually according to them, support from donor nucleic acid the ability such as the genetic expression of donor gene group, select recipient cell.This paper provides the transfer bacterial genomes to enter eucaryon host as illustrative methods, and it is not intended as restrictive.For example, bacterial genomes be transferred to there is preferred genetic operating system eukaryotic host cell (for example, yeast), in and after modifying therein, for the genomic expression gene product from modifying, it is necessary that genome is transferred back to the bacterial receptor cell.As discussed herein, the difference of translating and transcribing and the application of different codon etc. factor can stop expresses the donor gene product in host cell.Therefore, recipient cell can be mutually of the same race or sibling species with donorcells or biology.Order or boundary that it is normally identical with donor.The technician can, based on expectation from donor gene group or other nucleic acid of its expression, determine suitable recipient cell.
Donor nucleic acid is after separating in the agarose inserted block, optionally removes host DNA (for example,, by digestion and/or electrophoresis) and optionally uses methyltransgerase and/or protease treatment.
Can melt the agarose inserted block by incubation together with β-gelase I (New England Biolabs) for example, as embodiment 3A (ii) below (b) described in.
Can such as existing in situation, PEG-6000 or PEG-8000 or other PEG be shifted to be conducive to transform at polyoxyethylene glycol (PEG).The source of PEG, quantity and variable size are to determine optimum PEG.In one embodiment, PEG is PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG-10000, PEG-20000 or other.The concentration of PEG can change according to the condition shifted; Concentration for example comprise for or be approximately 5% for or be approximately 10% those.Example be described in following embodiment 3A (ii) (c) in.The inserted block melted can add recipient cell under there is situation in PEG, and gentleness shakes to mix.Allow to reclaim cell, centrifugal and grow in the substratum that comprises suitable selection substratum, with the recipient cell to comprising transfer donator nucleic acid, selected.On the one hand, cell is grown until colony occurs on substratum and under the condition suitable for the recipient cell type by bed board.Select colony, and continue selecting further growth on substratum, to produce the recipient cell that comprises metastatic gene group or other donor nucleic acid of desired amount.
The specific ratios that can keep as required recipient cell and donor nucleic acid.In one embodiment, can remain on every 2 μ g genomic dnas is or is approximately 10 7Individual and for or be approximately 10 8The ratio of the recipient cell between individual.The transfer method provided can be used for 200ng native gene group DNA is obtained to about 30 transformant, or each reaction shifts between bodies 500 and 1500, or other appropriate vol that obtain from host or donorcells.In a kind of non-limiting example, shift and to use for approximately 10 7In the agarose inserted block that comprises 100ng/ μ l donor gene group that individual recipient cell, 20 μ l melt, carry out.The ratio that should be appreciated that recipient cell and donor nucleic acid can change according to cell type, and the experience assessment can be used for optimizing this ratio.
Exemplary transfer method is illustrated in Fig. 8 (" 3 "): in the method, comprise appropriate flags and for the genomic dna of the element of breeding at host and recipient cell, separate from host cell in the agarose inserted block, with the methyltransgerase of crude extract or purifying, methylate and use the Proteinase K Deproteinization.Melt the agarose inserted block, by DNA incubation together with recipient cell, its then bed board selecting on substratum.As embodiment, the complete full mycoplasma mycoides LC donor gene group DNA that comprises YCp element, tetracycline marker and beta-galactosidase gene can separate in the agarose inserted block from yeast host, methylate with mycoplasma mycoides LC crude extract, and then use the Proteinase K Deproteinization.The agarose inserted block that melting comprises the genomic dna that methylates and together with the mycoplasma capri recipient cell incubation, and then bed board at the SP4 substratum that comprises the tsiklomitsin for selecting transformant.
G. alternative manner (repetition methods, iterative method)
Method described herein can be modified for many wheel genomes or other nucleic acid in the mode of iteration.
In one embodiment, the cell of transforming by the method (such as method by providing for example modified transfer in host cell, the recipient cell of modifying factor group are provided) can be used as the source of donor nucleic acid in the conversion with trailing wheel, modification and transfer, thereby generates genome and the biology of further modifying.
In a kind of non-limiting example, as institute's diagram in Fig. 1, the transformation bacterial cell of producing by the method (such as for example modified transfer in yeast of method by providing being provided, modifying the recipient cell of bacterial genomes) can be used as the source of donor nucleic acid in the conversion with trailing wheel, modification and transfer, thereby generates genome and the biology of further modifying.
Enter host cell in optional mobile donor gene group, transform it and shift by genome and make during it installs back the iteration embodiment of donor, by transforming, host's carrier to be inserted in the donor gene group.This genome is cloned into host cell; After the clone, any combination that all technology of use host genetic method are manufactured insertion, disappearance, rearrangement or modified in the donor gene group.This modifying gene group is followed separated and is transferred to recipient cell to generate the recipient cell that changes phenotype that has of transformation.Before shifting it, the donor dna that methylates is necessary to protect it to avoid recipient cell restriction system (one or mores') impact.Can start to repeat this circulation from the genome of new transformation.
Provide mobile bacterial genomes and enter yeast, transform it and shift and make it install back the non-limiting example of bacterium by genome in Figure 16, by transforming, yeast vector is inserted to bacterial genomes.This genome is cloned into yeast; After the clone, any combination that all technology of use yeast genetic method are manufactured insertion, disappearance, rearrangement or modified in bacterial genomes.The genome of this transformation is followed separated and is shifted and to enter recipient cell to generate the transformation bacterium.Before transfer, the donor dna that methylates is necessary to protect it to avoid the impact of the restriction system (one or more) of recipient cell.Can start to repeat this circulation (dotted arrow) from the genome of new transformation.
For many available, checking, confirmation and the reliable selective marker of selection and anti-selection yeast mutants, can use the method provided to carry out the seamless nucleic acid change of many for example unlimited iteration rounds at yeast host cell.Continuous modification to other unmanageable genomes or other large nucleic acids clone copy can in extremely rapid succession be carried out in yeast.The mating ability of yeast is favourable for modifying factor group and other large nucleic acids.When yeast activated between mating season, yeast restructuring machine can be used for generating library, for example, and the combinatorial library that comprises colone genome or nucleic acid variant.Although embodiment described herein utilizes yeast cell as host cell, but be to be understood that provided method comprises other host cells, described other host cells just for example are understood with the anti-selective marker aspect of mutant host cell body of selecting for selecting, or have been evaluated with regard to this point.Consider many modification of this embodiment and drop in the application's scope.
The application of the method and composition H. provided
The method and composition provided can be used for the problem that solution is relevant to environment, power generation and medical science.The method and composition provided can be used for production, transformation and modifying factor group and biological and for other products of commercial use, such as immunogen, bioprotein and chemical, vaccine, biofuel and useful protein such as enzyme.For example, the method provided can be used for operation and transformation particularly has those biological nucleic acid of weak genetic system from any biology, those that are not easy to operate by ordinary method such as its genome.The method provided can be used for building the synthetic gene group and shifts this genome and enters recipient cell to produce synthetic cell.Therefore, the method can be used for producing medically useful protein, comprises enzyme, albumen and exonuclease treatment agent, antibody, immunogen, vaccine and other cellular product.
Vaccine refers generally to improve the immunogenic formulation to the specified microorganisms with disease-related (bacterium or virus) immunity.Vaccine typically comprises the reagent of a small amount of similar microorganism.This reagent stimulates the immunity system of body take to identify this reagent as exotic, destroy it and remember it, so that any such microorganism that it runs into subsequently can more easily be identified and destroy to immunity system.Vaccine can be preventative (for example, prevention or improve by any natural or effect that " wild " pathogenic agent infects in the future) or curative (for example, cancer vaccine).Vaccine can be (multivalent) ((polyvalent) of also claiming multivalence) of unit price (also claiming monovalence) or multivalence.Univalent vaccine be can design and single antigen or single microorganism resisted with immunity.Can design multivalence (multivalent) or multivalence (polyvalent) vaccine resists two or more same microorganism bacterial strains or resists two or more microorganisms with immunity.In some cases, univalent vaccine can be used for the immunne response that fast Development is strong.Vaccine is for attempting to reduce the risk of disease, and the ability of useful immunne response is induced in reservation.Vaccine can comprise biology dead or inactivation or come from their purified product.The immunogenicity component can be used for processing the mankind and non-human colony (for example, primates, veterinary animal etc.).
The technology provided can be used for producing immune component, with from biology, such as the immunogenicity component, such as comprise viable cell and virus those, cause immunne response, described viable cell and virus include but not limited to, the Adenoviridae (Adenoviridae) (for example, adenovirus) of modifying, Picornaviridae (Picornaviridae) (for example, Coxsackie virus, hepatitis A virus, the vertebra poliovirus), herpetoviridae (Herpesviridae) (for example, various herpes simplex virus types, dust bar virus, human cytomegalic inclusion disease virus), Hepadnaviridae (Hepadnaviridae) (for example, hepatitis B virus), flaviviridae (Flaviviridae) (hepatitis C virus, yellow fever virus, dengue fever virus, west nile virus etc.), Retroviridae (Retroviridae) (for example, human immunodeficiency virus (HIV)), orthomyxoviridae family (Orthomyxoviridae) (for example, influenza virus), Paramyxoviridae (Paramyxoviridae) (for example, Measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, people's Metapneumovirus etc.), papillomavirus section (Papillomaviridae) (for example, papillomavirus), Rhabdoviridae (Rhabdoviridae) (for example, rabies virus), Togaviridae (Togaviridae) (for example, rubella virus) and Parvoviridae (Parvoviridae) (for example, human bocavirus (Human bocavirus), assays for parvovirus B 19), influenza (influenza) (for example, H1N1 influenza, Type B hemophilus influenzae (Haemophilus influenzae type B) etc.), poliomyelitis, cowpox, varicella zoster, reovirus (reovirus), retrovirus, poxvirus, parvovirus, picornavirus, paramyxovirus and BCG.
The technology provided can be used for producing immune component, with from biology, such as the immunogenicity component, such as comprising those of viable cell and bacterium, cause immunne response, described viable cell and bacterium include but not limited to the special Pseudomonas (Bordetella) (for example, Bordetella pertussis) of the Boulder of modifying, Borrelia (Borrelia) (for example, Borrelia burgdoyferi), Brucella (Brucella) (for example, alcaligenes abortus, the dog Brucella, brucella melitensis and brucella suis), campylobacter (Campylobacter) (for example, campylobacter jejuni), chlamydiaceae (Chlamydia) (for example, Chlamydia pneumoniae, ornithosis virus and chlamydia trachomatis), fusobacterium (Clostridium) (for example, Clostridium botulinum, clostridium difficile, clostridium perfringens and clostridium tetani), Corynebacterium (Corynebacterium) (for example, corynebacterium diphtheriae), enterococcus spp (Ehterococcus) (enterococcus faecalis and faecalis), Escherichia (Escherichia) (for example, intestinal bacteria), Mark Lewis-Francis Pseudomonas (Francisella) (for example, francisella tularensis), Haemophilus spp (Haemophilus) (for example, Haemophilus influenzae), Helicobacterium (Helicobacter) (for example, helicobacter pylori), Legio (Legionella) (for example, Legionella pneumophila), leptospira (Leptospira) (for example, leptospira interrogans), listeria (Listeria) (for example, listerisa monocytogenes in mjme), Mycobacterium (Mycobacterium) (for example, Mycobacterium leprae and mycobacterium tuberculosis), Mycoplasma (Mycoplasma) (for example, mycoplasma pneumoniae), neisseria (Neisseria) (for example, neisseria gonorrhoeae and Neisseria meningitidis), Rhodopseudomonas (Pseudomonas) (for example, Pseudomonas aeruginosa), rickettsiae (Rickettsia) (for example, Li Keshi rickettsia), Salmonella (Salmonella) (for example, Salmonella typhi and Salmonella typhimurtum), Shigella (Shigella) (for example, shigella sonnei (Shigella sonne)), Staphylococcus (Staphylococcus) (for example, streptococcus aureus, streptococcus pneumoniae, staphylococcus epidermidis and staphylococcus saprophyticus), suis (Streptococcus) (for example, streptococcus agalactiae, streptococcus pneumoniae and streptococcus pyogenes), treponema (Treponema) (for example, Treponoma palladium), Vibrio (Vibrio) (for example, vibrio cholerae) and yersinia's genus (Yersinia) (for example, yersinia pestis), it has the immunogenicity feature, makes them become attractive vaccine candidate.
Available vaccine preparation method can not remove the pathogenic immunogenicity that simultaneously keeps them of many biologies effectively.The method and composition that can be used for transformation and operation large nucleic acids and gene provided for example can be used for transforming such vaccine in combination.
Method described herein can be used for producing effective composition with treatment, prevention or significantly reduce following biotic influence: varicella, zoster (shinges), influenza, poliomyelitis, measles, parotitis, rubella, toxic shock, cholera, glandular plague, hepatitis A, hepatitis B, hepatitis C, yellow jack, malaria, tuberculosis, tetanus, encephalitis, acquired immune deficiency syndrome (AIDS) (AIDS), leprosy, canine distemper, canine parvovirus, infectious canine hepatitis, adenovirus-2, leptospirosis, Bordetella (bordatella), canine parainfluenza virus, singapore hemorrhagic fever, Lyme disease and other other diseases, for them, vaccine is useful in treatment and/or in processing one or more symptoms.
During other Virus Type, family and the relative disease that consideration is used in method described herein is provided at following table.
Figure BDA00002358547600561
Figure BDA00002358547600571
During the other bacterial species that consideration is used in method described herein and relative disease are provided at following table.
Figure BDA00002358547600572
Figure BDA00002358547600581
Figure BDA00002358547600591
Method described herein can be used for producing the composition of effective treatment or prevention contagious bovine pleuropneumonia (CBPP), and this disease is caused by bacterium mycoplasma mycoides microcolony.This disease, also referred to as pleuropneumonia, is the main pathogens of ox, farm cattle, buffalo and zebu.This disease in Africa, the Middle East, southern Europe and Asia Desk region-by-region wide-scale distribution.Therefore really need improved vaccine.This disease biology is the phylogeny close relative of bacterium---the large colony bacterial strain of mycoplasma mycoides GM12---who is used for illustrating the aspect of method provided by the present invention herein.The antigen gene of mycoplasma mycoides microcolony bacterium and/or genome can be used provided technology to clone and operate, for example, to have generated the cell of living vaccine effect, mutant.
The method provided is used together with the kind that is closely related with for example mycoplasma mycoides LC, as the pathogenicity bo for probing into mycoplasma and biological model system.The mycoides group of mycoplasma causes most of disease of ruminating animal, in the urgent need to vaccine.The method provided can be accelerated the structure of living vaccine bacterial strain.Method also can be used for determining that the minimum gene of life needs is supplementary, especially the minigene group such as the mycoplasma mycoides genome in.
The method of using vaccine is known in this area, and comprises that injection and aerosol delivery immunogenicity vaccine composition are to the experimenter.But compositions formulated is also used together with pharmaceutically acceptable carrier or vehicle.The composition that comprises the immunogenicity vaccine also is provided here, and described vaccine is formulated in the medicine that is used for the treatment of disease as herein described or situation.After being applied to the experimenter, can measure with regard to one or more aspects of immunne response the effect of immunogenicity vaccine composition.Immunne response comprises, for example induces antibody response (increase antibody titer), cytokine, induces T to assist (T H1 and T H2) cytodifferentiation and propagation etc.Replying that each is such can be quantized.Immunne response also comprises total bacterium or the virus load reduced in the patient.
Current disclosed method also can be used for developing biofuel.
Biomass crude oil (biocrude) is the mixture of biogenic compound or biogenic different compounds, as the raw material of refinery, replaces or supplementary crude oil or other forms of oil.Usually, but optional, these raw materials are by biological, chemistry, machinery or thermal treatment, to become the liquid state that is applicable to introducing refinery.
Microorganism can be used method described herein to modify to produce biomass crude oil, and it can be followed and further be processed into biofuel composition.This biofuel can then be used as finished product fuel or fuel dope.
" finished product fuel " refers to the mixture (producing by chemistry, thermochemistry or biological approach) of compound or chemical compound, and it is used directly as pure fuel or the fuel dope in engine in suitable chemistry and physical condition.In many cases, but not always, it is definite that the suitability of the finished product fuel used in engine application needs the specification of the essential physics and chemistry character that meets by description.Some examples of engine are: oil engine, gas turbine, steam turbine, external combustion engine and steam boiler.Some examples of finished product fuel comprise: the diesel oil used in ignition (diesel engine) oil engine, the rocket engine fuel used in aeroturbine, in boiler, use with the oil fuel of generating steam or the oil fuel used in external combustion engine, the ethanol used in the fuel flexible engine.The example of fuel specification is the ASTM standard of mainly using in the U.S. and the EN standard of mainly using in Europe.
" fuel dope " refers to the compound or the composition that use due to a variety of causes and another kind of fuel fabrication, the consumption of the product that described reason includes but not limited to meet the task of using biofuel, reduce the fossil oil source or the performance of enhancing fuel or engine.For example, fuel dope can be for changing zero pour/jellying point, cloud point, oilness, viscosity, oxidative stability, ignition characteristic, octane value and flash-point.Additive also can be used as antioxidant, emulsion splitter, oxygenation agent (oxygenates), the thermostability modifying agent, cetane number improver, stablizer, cold flow improver, combustion modifying agent, defoamer, fog dispersal additive (anti-haze additives), inhibitor freezes, the oil thrower cleaning additive, smoke suppressant, drag reducing additive, metal passivator, dispersion agent, sanitising agent, emulsion splitter, dyestuff, mark, static inhibitor (static dissipaters), sterilant and/or corrosion inhibitor.
70% oils of synthetic nearly its dry weight of some Eukaryotic Algaes.For these oils of photosynthate are desirable biofuel material standed fors.The biology that produces these oils can be grown in pond and desert, therefore for producing biofuel, can not lose tillable arable land.The slow growth that the use of these algae usually is subject to them limits.For example, but the method provided can be used for operating biological genome, transforms new biology, prokaryotic organism for example, it expresses the enzyme of participation oil route of synthesis, for example, by operation transcripting promoter, translation signals and codon optimized.The genome that the method can be used for modifying photosynthetic bacteria is to transform new bacterium, and it has the mosaic gene group of generation biofuel such as the oils produced by algae rather than photosynthetic normal product (glucose).
Other sugar that can make glucose and come from wood fiber biomass that use the recombinant microorganism of disclosed method manufacture can comprise transformation are converted into the biosynthetic pathway of Geraniol.
The recombinant microorganism (for example, the photosynthetic microorganism bacterial strain) that uses disclosed method to manufacture can be used for biology and produces branched-chain alcoho, for example comprises 2-methyl-1-butene alcohol, 3-methyl-1-butanol and isopropylcarbinol.Relate in one aspect to by introducing the heterologous gene of codase, produce the restructuring photosynthetic microorganism, described enzyme strengthens production and the decarboxylation of 2-ketone branched acids, causes producing corresponding branched chain aldehyde.Can then carry out the introducing of other gene, for effectively making branched chain aldehyde, be reduced into corresponding branched-chain alcoho.In addition, can transform microorganism, so that branched-chain alcoho is dewatered to produce various side chain alpha-olefins by enzymatic in vivo.
The recombinant microorganism coded plant fatty acyl-acp thioesterase that uses disclosed method to manufacture.Nucleic acid molecule can be used for inverting biological like this, such as photosynthetic organism and prokaryotic organism, for the synthesis of lipid acid and fatty acid product such as alkanoic, fatty alcohol, the fatty ester that comprises wax ester and hydrocarbon.Also comprise the biology that uses method provided herein to transform.
(for example use the recombinant microorganism of disclosed method manufacture; the restructuring photosynthetic microorganism) comprise nucleic acid molecule; it comprises that at least one produces the recombinant expression system of at least one external source fatty acyl-acp thioesterase; wherein said fatty acyl-acp thioesterase discharges the fatty acid chain that comprises 6-20 carbon, and the lipid acid that microorganism secretion is discharged by fatty acyl-acp thioesterase enters substratum.Thioesterase can be used for discharging the fatty acid chain that comprises 6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 carbon.The lipid acid so obtained can be by further synthesis modification or directly as the composition of biofuel or chemical.
In such structure, can expect to remove the Gene Partial in coding plastid transit peptides zone (plastid transit peptide region), because this zone is inappropriate in prokaryotic organism.Alternatively, if express, occur in eukaryotic cell, the plastid transit peptides coding region that host living beings is suitable can be replaced.Depend on the host, also can adopt preferred codon.
Further the genome of modified microorganism is to comprise that described synthetic enzyme preferably produces the acyl-acp with medium chain degree for the expression system of the heterologous gene of the β-one acyl synthetic enzyme (KAS) of encoding.This KAS enzyme can be used as increasing the operability for the acyl-acp molecule by the allos suitable length that medium chain acyl-ACP TE identifies and cuts.Another kind of example is that the photosynthetic host cell that comprises allos acyl-acp TE gene can further be modified the expression system comprised for one group of heterologous gene of the various subunits of the heterologous gene of multifunctional coded acyl group-CoA carboxylase or the acyl group of many subunits type of encoding-CoA carboxylase.The other enzyme of coding fatty acid biosynthetic pathway or other heterologous genes of composition also can be introduced and express in the host cell that comprises acyl-acp TE.
Also can modify photosynthetic microorganism so as one or more genes of coding β-oxidation path enzyme by inactivation or downward, or enzyme itself can the suppressed lipid acid degraded to prevent from discharging from acyl-acp, therefore increases the output of the lipid acid of secretion.In the situation that the product of expectation is medium chain fatty acids, inactivation or lower tone coded these chain lengths of preferential use are useful as the acyl group of substrate-CoA synthetic enzyme and/or the oxidasic gene of acyl group-CoA.The sudden change of medium chain specificity acyl group-CoA synthetic enzyme and/or medium chain specificity acyl group-CoA oxidase gene of encoding reduces the activity of enzyme, for the output that increases secretion lipid acid, is also effective.Modification in addition makes acyl-acp synthetic enzyme inactivation or downward or makes gene or protein inactivation.
Photosynthetic microorganism also can be modified, so that one or more genes of coding storage carbohydrate or polyhydroxyalkanoate (PHA) biosynthetic pathway enzyme are by inactivation or downward, or enzyme itself can be suppressed.Example comprises participation glycogen, starch or the synthetic enzyme of chrysolaminarin, comprises glucan synthase and q enzyme.Other examples comprise that the biosynthetic enzyme of participation PHA is such as acetoacetyl-CoA synthetic enzyme and pha synthesizing enzyme.
Disclosed method also can be used for producing industrial enzyme and industry is biological.Disclosed method can be used for producing the new biology with mosaic gene group, for example, for clostridium acetobutylicum (Clostridium acetobutylicum) and the chimeric genome of Clostridium cellulolyticum (Clostridium cellulolyticum), it has the gene from last kind, the coding enzyme that synthesizing alcohol needs from glucose; With the gene from a rear kind, coding is the cellulase of degraded cellulose effectively.Therefore, the method and composition provided can be used for producing effective degraded cellulose to produce the Cell and organism of ethanol.
Other are applied in and the following describes and be considered by this paper.The method generally can be used for cloning full genome and portion gene group, and it helps research from the genome of the biology that is difficult to cultivate and helps to build and breed synthetic genome.Although this paper has described some preferred industrial application, Method and process of the present invention is the extensive applicable instrument of any interested phenotype or product that produces from the genome of transformation.
I. embodiment
The embodiment that provides following embodiment to be provided to illustrate, and do not intend limiting the application's scope.
Embodiment 1
Bacterium donor gene group is transferred to yeast host cell
This embodiment has described and has used three kinds of different cloning process provided herein (Fig. 2) successful cloning bacteria genome in yeast host cell.As described below, every kind of method produces the host cell with nucleic acid, and described nucleic acid comprises the donor bacterium genome be connected with the yeast host carrier.By these methods, the donor gene group being transferred to host cell can use with together with the institute supplying method, in host cell propagation with modify the donor gene group, and enters recipient cell (Fig. 1) from host cell transfer donator genome.
By the yeast host carrier being inserted to then transformed yeast of donor bacterium genome, connect molecule thereby produce, connect molecule and then be transformed into yeast, the first cloning process that carries out showing in Fig. 2 A.Respectively by using homologous recombination to connect donor bacterium genome and yeast vector, second and the third method of carrying out showing in host cell in Fig. 2 B and 2C.Utilize the second method, bacterial genomes and yeast (host) carrier is dissolved yeast host cell (Fig. 2 B) by corotation.Host's carrier be comprise with bacterium donor gene group in the linear yeast vector of stub area of site homology, thereby and be inserted into by homologous recombination.Utilize third method (Fig. 2 C), a plurality of overlapping fragmentses of bacterial genomes are transformed into yeast host cell together with yeast vector.Homologous recombination in yeast cell realizes homologous recombination, and junction fragment comprises the genomic molecule of the donor bacterium be connected with yeast vector (Fig. 2 C) with yeast vector with generation.Adopt the research of each method to be discussed in more detail below.
Utilize each method, the mycoplasma genitalium genomic clone is entered to yeast host cell.In addition, use the first method, mycoplasma mycoides LC and mycoplasma pneumoniae genome are transferred to yeast, described at Fig. 2 A.Use the first method (Fig. 2 A), mycoplasma genitalium further is transferred to single yeast cell with mycoplasma mycoides LC genome as the molecule separated (each is connected with the yeast host carrier).
Example I A
Use the yeast vector of integrating that the full genome of mycoplasma donor is transferred to yeast host cell
This embodiment has described and has used the first for the donor gene group being introduced to the method for host cell, successfully three kinds of different mycoplasma donor gene group (mycoplasma genitalium bacterial strain MS5 of clone's (shifting and propagation); Mycoplasma mycoides subspecies mycoides, large colony bacterial strain GM 12; With mycoplasma pneumoniae bacterial strain M129-B170 (ATCC 29343)) enter host's yeast cell.Mycoplasma genitalium bacterial strain MS5 is the derivative of mycoplasma genitalium G37 (GenBank L43967).It is manufactured by interrupting gene in the G37 bacterial strain, as at Dhandayuthapani etc., in J Bacteriol 183,5645 (2001), describes.Mycoplasma mycoides subspecies mycoides, large colony bacterial strain GM 12 (genome sequence with Genbank accession number NZ_AAZK01000004.1 (GI:149364882)) is described in DaMassa etc., Am J Vet Res 44,322 (1983) and Lartigue etc., in Science 317,632 (2007).Mycoplasma pneumoniae bacterial strain M129-B170 (ATCC 29343) is the derivative of mycoplasma pneumoniae M129, GenBank accession number U00089.2 (GI:26117688).
Shifted each mycoplasma donor gene group by yeast vector being inserted to the donor gene group, the nucleic acid molecule that comprises genome and host's carrier with generation, and then this molecule is introduced to host cell by conversion.
I. build the three host's carriers that shuttle back and forth and enter yeast for cloning the mycoplasma genome
Design two the three yeast host carriers that shuttle back and forth, so that be used in the method shown in Fig. 2 A, clone's mycoplasma genome enters yeast host cell.These carriers---pmycYACTn and miniTn-Puro-JCVI-1.7, schematic illustrations is in Fig. 3.
A. build the pmycYACTn carrier
Carrier pmycYACTn (Fig. 3 A) length is 10kb and comprises: (i) from the height of pUC19 copy initial point (ori) with for amicillin resistance mark, (ii) the IS256 transposase gene of breeding intestinal bacteria with enter mycoplasma genome inverted repeat for swivel base; (iii) tetM and lacZ mark, the two is by the spiralin promoter expression, and as at Lartigue etc., J Bacteriol 164,1094 (1985) is described, for selecting and examination intestinal bacteria and mycoplasma; (iv) autonomously replicating sequence (ARS) and centromeric sequence (CEN), for copying at yeast and separating; (v) HIS3, select yeast selectable marker.
Overlapping fragments from be illustrated in Fig. 3 E (being labeled as fragment 1,2 and 4-7) carrier construction, it uses disclosed external assembly method (Gibson etc., Science 319,1215 (2008), U.S. Patent Application Serial 12/247,126 and WO09/048885, all this paper that is incorporated to by reference).Fragment 1 (1846 base pairs (bp)) comprises intestinal bacteria amicillin resistance (bla), pUC19 initial point, and it is included to promote the high yield plasmid to separate.Fragment 2 (1256bp) comprises mycoplasma IS256 transposase and promotor and IS256 inverted repeat (being labeled as " 3 " in Fig. 3 E), and it is included to promote that the carrier by swivel base inserts.Each comprises fragment 4 (2294bp) and 5 (3335bp) mycoplasma spiralin promotor and comprises respectively mycoplasma tetracyclin resistance (tetM) and LacZ gene, and it is included to promote that carrier is inserted to the donor gene group to be selected.Fragment 5 comprises the IS256 inverted repeat in addition to promote swivel base to insert.Fragment 6 (847bp) comprises yeast saccharomyces cerevisiae HIS3 gene and promotor, and it is included to promote being transformed into the selection of host cell.Fragment 7 (505bp) comprises yeast saccharomyces cerevisiae ARSH4 and CEN6 gene, and it is included to promote copy and separate.See Fig. 3 E.
By PCR, the primer of listing in use table 1 (Integrated DNA Technologies, Coral ville, IA) builds these overlapping fragmentses.In table 1, the zone overlapping with other fragments underlined; IS256 inverted repeat (being labeled as " 3 " in Fig. 3 E) is the black matrix type.Following plasmid is used as template in the PCR of independent fragment.For fragment 1,4 and 5, template is the pBS+ part (Stratagene, San Diego, CA) from the pMYCO1PSlacZ plasmid of pMYCO1 plasmid modification, be described in (2003) such as Lartigue and Blanchard, Nucleic Acids Res 31 (22): in 6610-8.For fragment 2, template is the 3.7kbPcil-Sal1 fragment of pISM31.1 carrier, is described in Pour-El etc., in plasmid 47 (2): 129-37 (2002).For fragment 6 and 7, template is the pARS-VN plasmid, is described in Noskov etc., and BMC Genomics 4 (1): in 16 (2003).
Table 1: for the PCR primer of the fragment that builds intestinal bacteria-mycoplasma-yeast shuttle vector pmycYACTn
Figure BDA00002358547600641
In order to produce every bar segment (amplicon), use the plasmid template of 10ng to carry out PCR in 100 μ L reaction volumes.Comprise the primer of pointing out, Phusion archaeal dna polymerase, HF damping fluid (New England Biolabs, Ipswich, MA) according to manufacturers's scheme amount in table 1, add in addition MgCl 2Making ultimate density is 2.0 or 3.0mM.Cycling condition is as follows: 98 ℃, and 30 seconds; Then 98 ℃ of lower incubations 10 seconds, anneal 30 seconds and in 30 circulations of 90 seconds of 72 ℃ of incubations; Then 72 ℃, 5 minutes.Between circulation and the annealing temperature between different fragments PCR can change as follows.For circulation 1-5, annealing temperature, between 46 ℃ and 59 ℃, increases by 5 ℃ (between 51 ℃ and 64 ℃) for circulation 6-30.Particularly, circulation 1-5 annealing temperature is 1,56 ℃ of fragment and 59 ℃; 5,46 ℃ and 48 ℃ of fragments; 4,46 ℃ and 50 ℃ of fragments; 2,46 ℃ of fragments; Fragment 6 and 7,48 ℃ and 52 ℃.For circulation 6-30, each temperature is higher 5 ℃ than the temperature of circulation 1-5.For each fragment, concentrate the PCR product and use β-gelase (New England Biolabs, Ipswich, MA) gel-purified amplicon.
For the fragment 2 that comprises transposase gene, from template pISM31.1, increase, one of PCR primer is included in 20 base pairs of standard of desired location and template homology, but also comprises and 2 of the IS256 inverted repeat 26 base pairs of copy homologies in addition, and it also is present in other parts of plasmid.In order to be conducive to the specific amplification of correct fragment, two restriction enzyme digestion of Pcil and Sail for these IS256 copies, with the template part of pISM31.1 expectation, separate, the 3.7kb fragment that then sepharose-purifying correctly obtains, it then is used as template in the pcr amplification of fragment 2.
Use disclosed external assembly method, the fragment of assembling purifying, generate the pmycYACTn carrier, described external assembly method is described in D.G.Gibson etc., Science 319, in 1215-1220 (2008), U.S. Patent Application Serial 12/247,126 and WO 09/048885, all this paper that is incorporated to by reference.As described (Gibson etc., Science 319,1215-1220 (2008); U.S. Patent Application Serial 12/247,126; With WO 09/048885), by having 5% PEG-8000,50mM Tris-Cl, pH 7.5,10mM MgC1 2, 10mM DTT, the every dNTP of 25ug/ml BSA, 200uM and 1mM NAD situation under, this assembling and Taq DNA ligase and Taq archaeal dna polymerase, 45 ℃ of lower incubations 15 minutes, are repaired to whole " chewing rear assembling (chew back assembling) " and (CBA) reacted.Reaction is then carried out phenol extraction, isopropanol precipitating, resuspension electroporation and is entered EPI300 cell (Epicentre Biotechnologies, Madison, WI).Select transformant with Pyocianil.Use three restriction digestion that separate, the plasmid of the correct size of screening from the DNA that selects the clone.As follows, the existence of the various elements of test plasmid on phenotype.In intestinal bacteria, propagation is guaranteed the function of pUC19 initial point; Select with Pyocianil and tsiklomitsin in intestinal bacteria and use the X-gal screening verification to have the complete copy of bla, tetM and lacZ mark; With show HIS3, ARSH4 and CEN6 mark with the successful transformed yeast of the carrier that separates and can use.As following, describe, by being transformed into mycoplasma, confirmation has the existence of the transposon of function.
B. build the miniTn-Puro-JCVI-1.7 carrier
The long 14kb of miniTn-Puro-JCVI-1.7 carrier (Fig. 3 B), and identical with pmycYACTn except following: (i) it does not comprise lacZ; (ii) it comprises the tetracycline resistance marker, rather than the tetM mark; (iii) it comprises bacterial artificial chromosome (BAC) carrier.
Ii. host's carrier is inserted to the donor gene group
A. mycoplasma genitalium MS5
In preparation transfer mycoplasma genitalium bacterial strain MS5 donor gene group enters yeast host cell, by electroporation mycoplasma genitalium donorcells, carrier pmycYACTn carrier is inserted to the donor gene group, as at J.I.Glass etc., described in PNAS USA 103,425 (2006).By in the situation that exist tsiklomitsin to grow, select transformant, and select mono-clonal for further analyzing.Use the primer of carrier inside to carry out the site of direct gene group order-checking (as at J.I.Glass etc., PNAS USA 103,425 (2006) described in) to determine that carrier inserts.Sequence between two IS256 inverted repeats that the clone who selects comprises pmycYACTn also comprises two IS256 inverted repeats of pmycYACTn, and swivel base, (Fig. 3 C) have as designed occurred in indication.Transposase, pUC19 initial point and ampicillin resistance gene lose during swivel base.Host's carrier insert in the donor gene group in nonessential MG411 genome (J.I.Glass etc., PNAS USA 103,425 (2006); C.A.Hutchison etc., Science 286,2165 (1999)).
B. mycoplasma mycoides subspecies mcycoides, large colony bacterial strain GM12
Preparing to shift mycoplasma mycoides subspecies mycoides, large colony bacterial strain GM12 genome enters in yeast host, and the mycoplasma donorcells is used PEG to transform with pmycYACTn, as at K.W.King and K.Dybvig, described in plasmid 26,108 (1991).By being supplemented with growth selection transformant on the flat board of tsiklomitsin.
The site that the clone of four selections inserts the donor gene group by direct gene group sequencing analysis with location host's carrier.Result shows, in each of four clones, whole host range plasmid has been integrated into the donor gene group, rather than by swivel base integrated part pmycYACTn construct.In three of four clones, carrier (pmycYACTn) is inserted into by the exchange event (crossover event) in the pUC initial point or very contiguous pUC initial point place.In four clones, in yeast HIS3 gene, exchange has occurred, and therefore less than the transforming gene group for subsequently, entered yeast.The insertion of host's carrier has occurred in the position of adjacent I S1296 element in all cases.
The clonal growth of schematic illustrations in Fig. 3 D is sane.For the genome that confirms this clone can shift effectively, to carry out at C.Lartigue etc., the calcium chloride conversion process of describing in Science 317,632 (2007) is so that it is transferred to the mycoplasma capri recipient cell from the mycoplasma genitalium donorcells.Clone 1.1 genome effectively shifts and enters the mycoplasma capri host cell.Therefore, select this clone for from donor mycoplasma metastatic gene group, entering yeast host cell.
C. mycoplasma pneumoniae bacterial strain M129-B170 (ATCC 29343)
In preparation transfer mycoplasma pneumoniae bacterial strain M129-B170 donor gene group enters yeast host cell, mycoplasma transforms by electroporation with MiniTn-Puro-JCVI-1.7, as at J.I.Glass etc., described in PNAS USA 103,425 (2006).Select tetracycline resistance transformant storehouse in liquid culture.
Iii. separate and comprise the donor gene group of inserting yeast vector
For fracture is minimized, scheme according to manufacturers's suggestion, use low melting-point agarose and Bio-Rad CHEF mammalian genes group DNA inserted block test kit (Bio-Rad Laboratories, Hercules, CA), separate in the agarose inserted block and comprise the donor gene group that yeast vector inserts.Comprising the genomic agarose inserted block of the mycoplasma with insertion dialyses 1 hour to 10mM Tris pH 8.0 (using in some cases 1mM EDTA).Inserted block is then dialysed a few hours to 10mM (6%) PEG 6000 (United States Biochemical), 0.6M NaCl, as at S.Katsura etc., described in Electrophoresis 21,171 (2000).Separation is comprised to carrier and enter the mycoplasma mycoides LC genome (below seeing) of W303 cell for transfer, or separation is comprised to carrier enter the mycoplasma pneumoniae genome (see below) of VL6-48N cell for transfer, do not carry out this PEG/NaCl processing.
Inserted block then melts 5 minutes under 65 ℃, add thereafter 65 ℃ of TE of 2 volume and leniently stir mixture, and under 65 ℃ further incubation 5 minutes.Use 20 microlitres to be transformed.
The donor gene group that iv. will have yeast vector is transferred to yeast host cell
For the donor gene group is introduced to host cell, use N.Kouprina and V.Larionov, Nat Protoc3,371 (2008) the open methods of describing, except culture sometimes grows to the OD that is less than recommendation, use the DNA transformed yeast spheroplast from inserted block.
Use the method, all three genomes that will comprise the yeast vector inset are transformed into the yeast strain VL6-48N (V.Larionov etc., PNAS USA 94,7384 (1997)) be developed for high transformation efficiency.Mycoplasma genitalium cll6-2 and mycoplasma mycoides LC cl1.1 genome with inset also are transformed into W303a bacterial strain commonly used (MATa his3 leu2 ura3 trp1 ade2).Mycoplasma mycoides LC cll.l genome with inset also is transferred to the yeast strain of restructuring-defect, VL6-48-Δ 54G, its be defective in the RAD54 gene (MATa his3-Δ 200 trp1-Al ura3-52 lys2 ade2-101 metl4 rad54-Δ l::kanMX).Be transformed into VL6-48-Δ 54G bacterial strain to solve genome potentially unstable in other yeast---due to the restructuring between a plurality of almost identical 1.5kb IS1296 copies---possibility.In the RAD54 gene, the yeast strain of defect can reduce the generation (Y.Le and M.J.Dobson, Nucleic Acids Res 25,1248 (1997)) of many recombination event in yeast artificial chromosome (YACs).With the isogenic rad54 mutants which had of VL6-48N, be almost from Vladimir Larionov (Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health) present.
V. analyze and confirm that full genome shifts
Analysis is transferred to host cell from the DNA of each transformed yeast host cell to confirm the donor gene group that comprises complete host's carrier.The mycoplasma genome of cloning in yeast by multiplex PCR (MPCR) screening is to confirm integrity.Use the multiple PCR reagent kit from Qiagen (Valencia, CA), use 1 or 2 group, every group 10 amplicons, use the primer from IDT, carry out MPCR.Each reaction is described in greater detail below.
In order to confirm the genomic size that comprises inset, by gel electrophoresis, separate the also DNA of analyzing total from comprise the genomic single yeast clone of mycoplasma, as following, described.Usually, use the scheme DNA isolation from Bio-RadCHEF-DR III handbook " cerevisiae dna for preparing the agarose embedding ".Wherein indication, under constant voltage, prerunning inserted block several hours is to remove yeast chromosomal dna.Wherein indication, in order to increase the efficiency of this step, but at first inserted block digests with AsiSI, Fsel and the RsrII in cutting yeast chromosomal nonrecognition site in mycoplasma genitalium or mycoplasma mycoides LC.The DNA separated limits digestion (with the enzyme (one or more) of pointing out), then reverse electric field (Bio-Rad FIGE Mapper) or pulsed field (Bio-Rad CHEF-DR II or III system) electrophoresis, as indicated, or by heating linearizing 1 hour under 55 ℃.Wherein southern blotting technique is carried out in indication on gel.As at D.G.Gibson etc., Science 319, description in 1215 (2008), carry out southern blotting technique, except in some cases---probe mark and survey and use the direct mark of Amersham AlkPhos and detection system (the GE Healthcare with CDP-Star wherein, Piscataway, NJ).
A. from yeast host PCR, separate and analysis mycoplasma genitalium genome
From the mycoplasma genitalium cll6-2 genome that will comprise yeast vector transfer to reclaim after bacterial strain VL6-48N 24 separately the clone genomic dnas with from mycoplasma genitalium is transferred to after the W303a bacterial strain reclaim 8 separately clone's genomic dnas in the agarose inserted block, separate, and by pcr analysis to confirm integrity.Mycoplasma genitalium synthetic gene group (sMgTARBAC37, as the generation in (D.G.Gibson etc., Science 319,1215 (2008)) middle description) and natural mycoplasma genitalium genome are as positive control.
The PCR primer of listing is in the following Table 2 used to generate amplicon in the MPCR reaction, and it separates equably around the mycoplasma genitalium genome that comprises the yeast vector inset.Along the position display of the amplicon of genome length in Fig. 4 A, the size of black lines indication and digitized representation amplicon for amplicon wherein.Being summarised in table 5 for the result (data do not show) that reclaims the PCR of DNA from the clone at VL6-48N and W303.By pcr analysis (data do not show), 22 (22) individual and from 5 performances of 8 clones of W303a strains separation complete (for the product of the correct size of each amplicon) from 24 clones of 24 VL6-48N strains separation.
Table 2: mycoplasma genitalium multiple PCR primer order (organizing 1)
Figure BDA00002358547600681
Analyzing on Size
For the mycoplasma genitalium cll6-2 genome confirming to comprise yeast vector in the complete clone from the VL6-48 bacterial strain is correct size, to thinking that by MPCR complete 3 clones (11,16 and 24) carry out the CHEF gel analysis.For this process, use scheme from Bio-Rad CHEF-DR III handbook " cerevisiae dna for preparing the agarose embedding " in the agarose inserted block from the clone DNA isolation.In order to remove chromosomal DNA, inserted block under constant voltage prerunning several hours to remove yeast chromosomal dna.In order to improve the efficiency of this step, but at first inserted block digests with AsiSI, Fsel and the RsrII in cutting yeast chromosomal nonrecognition site in mycoplasma genitalium.
After prerunning, with EagI or BssHII DNA digestion.The genomic fragment of the mycoplasma genitalium with carrier inset produced by these enzymes and their size are pointed out in the collection of illustrative plates of Fig. 4 A and near hurdle collection of illustrative plates.The DNA of digestion is separated by the gel electrophoresis of reversing electric field (Bio-Rad FIGE Mapper).Result is summarised in (gel does not show) in table 5.Two (11 and 16) in these three clones are the sizes of expectation.
For the mycoplasma genitalium cll6-2 genome confirming to comprise yeast vector in the complete clone from the W303 bacterial strain is correct size, 5 complete clones are carried out to the CHEF gel analysis.For this process, by the top description to the VL6-48N bacterial strain, separate the DNA from these clones in the agarose inserted block, under constant voltage, prerunning several hours is to remove Yeast genome, as above-mentioned.The DNA separated then uses the EagI linearizing.
Separate sample by pulsed field gel electrophoresis.Synthetic sMgTARBAC37 genome (see above) cuts and is used as positive control with NotI.Result is summarised in (gel does not show) in table 5.4 in 5 clones is the size of expectation; Clone's 4 extra faint bands that comprise about 300kb.
B. by PCR, from yeast host, separate and analysis mycoplasma mycoides cl1.1
As above-mentioned, the genomic dna of 48 independent clone and separate that reclaim after the mycoplasma mycoides LC cI1.1 genome that will comprise the yeast vector inset is transferred to bacterial strain VL6-48N by multiplex PCR assay, to confirm integrity.Use from the scheme of Bio-Rad CHEF-DR III handbook " cerevisiae dna for preparing the agarose embedding " in the agarose inserted block from the clone DNA isolation.
The PCR primer that design is listed in table 3, and for generating amplicon to estimate the genomic integrity of mycoplasma mycoides shifted.As shown in Fig. 5, amplicon the majority of IS1296 element between, in Fig. 5, diagrammatic has on the genomic collection of illustrative plates of inset and is indicated by arrow.Other 230bp amplicon is the HIS3 marked region of yeast vector.Another primer sets (NSF1179/18 and NSR1642/16; In Table 3) produce the amplicon (zone of yeast saccharomyces cerevisiae rDNA) of 464bp, and in order to test as positive control.The size of primer sets and consequent amplicon is listed in table 3.
Be transformed into the mycoplasma mycoides LC cI1.l genome that comprises the yeast vector inset in Δ 54 restructuring-defect bacterial strains with multiplex PCR assay, obtain similar result (gel does not show).
The use by oneself DNA of the W303a cell that the mycoplasma mycoides LC cI1.1 genome that comprises the yeast vector inset transforms of multiplex PCR assay, show that 8 in 15 clones are complete (gel do not show).
Table 3: mycoplasma mycoides LC multiple PCR primer sequence
Figure BDA00002358547600691
Figure BDA00002358547600701
Analyzing on Size
For the mycoplasma mycoides cI1.1 genome confirming to comprise yeast vector in the complete clone from the VL6-48 bacterial strain is correct size, 6 complete clones are carried out to the southern blotting technique analysis of CHEF gel analysis, show that 5 in 6 are correct sizes.
3 that mycoplasma mycoides cI1.l genome with comprising yeast vector is transformed in these 5 clones that reclaim after the VL6-48N bacterial strain are carried out the CHEF gel analysis as follows: as mentioned above, in the agarose inserted block from clone (07,14 and 38) DNA isolation, without prerunning.The DNA separated digests with BssHII and follows by pulsed field gel electrophoresis (CHEF) separately.For each clone, analyze parental generation clone and 3-4 subclone (the gel result does not show).Mark and the contrast in analysis, used are: (1) hangs down scope PFG mark (New England Biolabs, Ipswich, MA); (2) yeast saccharomyces cerevisiae mark (Bio-Rad); (3) VL6-48N ((4) of indigested (3) and BssHII-digestion); With mycoplasma mycoides LC cI1.l ((6) of indigested (5) and BssHII-digestion).Result shows that all 3 clones are for correct size and stable.
Mycoplasma mycoides LC cI1.l genome with comprising the yeast vector inset is transformed to 8 complete clones that reclaim after the W303a cell and carry out the CHEF gel analysis to confirm size.For this process, as mentioned above, in the agarose inserted block, from clone and separate DNA, use as mentioned above prerunning to remove Yeast genome.DNA untreated (-) or with BssHII digestion (+).DNA then separates by pulsed field gel electrophoresis.Electrophoresis mycoplasma mycoides LC cI1.l DNA is as positive control.Result is summarised in (the gel result does not show) in table 5.Result means each genome that comprises correct size of 8 clones.
C. by PCR, from yeast host, separate and the analysis mycoplasma pneumoniae
The genome of 20 independent clone and separate that reclaim after the mycoplasma pneumoniae genome that will comprise yeast vector is transferred to bacterial strain VL6-48N, as mentioned above by multiplex PCR assay to confirm integrity.Carry out two different multiplex PCRs.List (the gel result does not show) for group 1 and the primer of group 2 and the size of consequent amplicon in table 4.As shown in Fig. 4 A, amplicon is (with black lines and numeral indication; The inside: organize 1; Outside: organize 2) around the mycoplasma pneumoniae genome that comprises the carrier inset, separate equably.Result shows that 13 of 20 transformant is complete.
Table 4: mycoplasma pneumoniae multiple PCR primer sequence
Figure BDA00002358547600711
Figure BDA00002358547600721
Analyzing on Size
9 to these complete transformant are carried out the CHEF gel analysis.For this process, use scheme from Bio-Rad CHEF-DR III handbook " cerevisiae dna for preparing the agarose embedding " in the agarose inserted block from these clones DNA isolation.In order to remove chromosomal DNA, inserted block under constant voltage prerunning several hours to remove yeast chromosomal dna.DNA is then with NotI or SbfI digestion.The genomic fragment of the mycoplasma pneumoniae with carrier inset produced by these enzymes (numbering 1-6) is pointed out in the collection of illustrative plates of Fig. 4 B, in fragment and near the hurdle of their size collection of illustrative plates, lists.
The DNA of digestion by pulsed field gel electrophoresis (Bio-Rad CHEF-DR II or III system) separately.Result is summarised in (the gel result does not show) in table 5.Restricted fragment as be numbered in Fig. 4 B.Yeast clone 8 is not by complete digestion (data do not show).
Table 5 is summed up the result from embodiment 1 (A), and 3 mycoplasma genomes that wherein comprise the yeast vector of integration are transferred to yeast.By the multiplex PCR with 1 or 2 group, every group 10 amplicons, screen complete clone.Digest and gel electrophoresis by restriction, follow in some cases southern blotting technique, test clone's size.
Table 5: 3 mycoplasma species genomes of the yeast vector that the clone comprises integration in yeast
Figure BDA00002358547600722
Figure BDA00002358547600723
Figure BDA00002358547600731
Example I B
By the full genome of homologous recombination and yeast host carrier, the mycoplasma genitalium genome is transferred to yeast host thin Born of the same parents
The method that use is described in Fig. 2 B, by the homologous recombination of genome in host cell and yeast vector, be transferred to yeast cell by linearity, the full genome of mycoplasma genitalium.The mycoplasma genitalium genome comprises 3 single point of contact restriction sites, wherein 2 rRNA operons that are positioned at it.The 3rd 3 ' end that is positioned at the tRNA encoding sequence.Because can design the clone to preserve the integrity of tRNA, by close this AscI site of homologous recombination insertion vector.
Yeast clone carrier pARS-VN is (at V.N.Noskov etc., BMC Genomics 4, description in 16 (2003)) be used as the template of PCR, use pair of primers, each comprise with the 60bp of the regional homology of insertion point flank in the mycoplasma genitalium genome and with the 20bp of carrier homology.Provide primer by IDT PAGE-purifying.Their sequence is as follows, and the carrier sequence is black matrix, and C1aI (the first primer) or XhoI (the second primer) site are for underlining:
TTAATAACAAAAAAATCTCTATTAAAAAAACCAACTTTAAAGTTGGTTTGAAATTC TAAA (SEQ ID NO:97) and
GGATAGAGTGTCTGGCTTCGGACCAGAAGGTTATGGGTTCAAGTCCTATTGGGCGCGCCA(SEQ?ID?NO:98)。
Figure BDA00002358547600733
Utilize the whole carrier of carrier pcr amplification of these primers, except the 9bp between single (unique) C1aI and XhoI restriction site, produce the 6.5kb product.
Prepare this linear carrier DNA and mixture from the DNA of mycoplasma genitalium bacterial strain MS5, for corotation, dissolve yeast strain VL6-48N spheroplast.Mycoplasma genitalium DNA separates as follows in the agarose inserted block, so that fracture minimizes.The mycoplasma genitalium genomic dna in the low melting-point agarose inserted block from the bacterial strain MS5 grown at the SP-4 substratum with two batches of separation.Supplement gentamicin to 200 μ g/ml for the culture of the 2nd batch.PBS rinsing twice for adherent cell, then scrape in the damping fluid that comprises 8.0mM HEPES, pH 7.4,272mM sucrose and 10% glycerine.Each inserted block comprises from about 6 (the 1st batches) or 10 (the 2nd crowd) cm 2The DNA of fused cell.For molten born of the same parents, by the cell in agarose under 50 ℃, the for some time be incubated overnight between 2 days in 0.4MEDTA, 0.4%N-Sarkosyl L and 2mg/ml Proteinase K, follow exchange buffering liquid, and process for the second time identical time range under identical condition.Inserted block is thoroughly dialysed to 10mM Tris, 50mM EDTA, then in the 10mM Tris, the 50mM EDTA that supplement PMSF to 0.1mM, dialyse 2 times, each 2 hours, and then dialyse again in 10mM Tris, 50mM EDTA and be stored in 10mM Tris, 50mM EDTA.
Before conversion, melt inserted block and digest with gelase, as follows.First inserted block is by CHEF (Bio-Rad) electrophoresis twice, and it removes broken DNA, and retains complete ring-type genome (to increase the complete genomic frequency of ring-type in inserted block).On 1% pulsed field sepharose, with carrying out the first electrophoresis the switching time of 0.5 * TBE and 50-90 second, over 20 hours.On 1% low melting point gel, with carrying out the second electrophoresis the switching time of 1 * TAE and 60-120 second, over 24 hours.Two gels are all under 14 ℃, with 120 °, 6V/cm electrophoresis.Each 3 inserted blocks from 2 batches are thoroughly dialysed to aseptic 1 * TAE, under 73 ℃, melt several minutes, and balance to 42 ° then digests 1.5 hours with β-gelase I (New England Biolabs, Ipswich, MA).Half of each volume moves in new Eppendorf pipe.Before conversion, genome uses 20U Ascl (in 1 * NEB damping fluid 4; New England Biolabs, Ipswich, MA) digest and spend the night under 37 ℃, it produces double-strand break near the site of intention and the restructuring of host's carrier, as institute's diagrammatic in Fig. 6 A.
For by the donor gene group (by with the yeast vector cotransformation) introduce host cell, use Kouprina and Larionov, Nat Protoc 3,371 (2008) the open methods of describing, except culture sometimes grows to the OD that is less than recommendation, use the DNA transformed yeast spheroplast from inserted block.Utilize the method for the disclosure, as above-mentioned, yeast cell is suspended in the 1M Sorbitol Powder and before transforming and uses Zymolyase TM(β-1,3-dextran kelp pentasaccharides lyase) processed to remove cell walls.The DNA reclaimed from agarose inserted block incubation together with spheroplast.After reclaiming in growth medium, the cell bed board is in selective medium.
Selected clone is also by multiplex PCR and gel electrophoresis assessment, as embodiment 1A (v) in the above (a) described in, except using two groups of 10 amplicons, rather than one group.For the primer that generates first group of amplicon in the above table 2 list.For generating the primer of second group of amplicon and the size of consequent amplicon is listed in the following Table 6.
Table 6: mycoplasma genitalium multiple PCR primer sequence (the 2nd group)
Figure BDA00002358547600741
Transform 45 transformant of generation with the DNA of AscI-digestion.All these use primer by the multiplex PCR inspection, to produce 20 amplicons, (table 2, table 6) as discussed above.21 performances are complete.Southern blotting technique by the CHEF gel checks this 21 transformant, and 15 show as correct size.Transform and produce 50 transformant with indigested mycoplasma genitalium genome.All these are by identical multiplex PCR inspection, and 7 performances are complete.One in these is correct size.These results show by with restriction enzyme recombination site digestion and with yeast host carrier cotransformation, then in yeast host cell by recombinating in body, the full genome of successful transfer donator mycoplasma.
Embodiment 1C
By recombinating in body, assemble genomic overlapping fragments and yeast host carrier in yeast host cell, The mycoplasma genitalium genome is transferred to yeast host cell
This embodiment has described and has used the method be illustrated in Fig. 2 C successfully shift mycoplasma genitalium donor gene group to yeast host cell and breed therein.In the method, by the genomic a plurality of overlapping fragmentses of homologous recombination, assembling genome in host cell (yeast).
The fragment (piece) that use comes from intestinal bacteria BAC clone carry out this strategy (see Gibson etc., Science319,1215 (2008), comprise supplementary at wire material; Gibson etc., PNAS USA, (2008) 105:20404-9; And U.S. Patent application, publication number 12/247,126, contriver's name: Gibson etc.).
In this research, use above-mentioned disclosed first method (at Gibson etc., Science 319, the multi-stage methods of describing in 1215 (2008) and supplement at wire material), by using vitro recombination and using the BAC carrier to clone in intestinal bacteria, by the assembling of three phases, at first generate 1/4 genome (each about 144kb), assemble synthetic mycoplasma genitalium genome from 6 fragments (piece).These 4 " 1/4 genome " (1-4) diagrams in Fig. 6 B.
As described, carry out isolation of genomic DNA from host cell, not prerunning and digest to exhaust pastoris genomic dna without the yeast specific enzymes in embodiment 1A.As above-mentioned, use CHEF to analyze, analytic sample.Also carry out southern blotting technique (data do not show).
Table 7: the genomic primer of mycoplasma genitalium of transferring to yeast cell for analyzing the homologous recombination of using overlapping fragments
Figure BDA00002358547600751
Figure BDA00002358547600761
Result shows that 6 of these transformant comprise correct sequence.Utilize indigested sample, only obtain 2 transformant, wherein without any one, in PCR and southern blotting technique analysis, show complete mycoplasma genitalium genome.Use as above-mentioned identical process is carried out another research, except Quarter 3 rather than AscI digestion in the cutting of the single BsmBI site of this Quarter.Use identical conversion and analytical procedure.Produce 73 transformant with the research of BsmBI digestion, when estimating by PCR wherein 44 be correct.28 these clones that check by southern blotting technique five (5) individual be correct.These results show when before transforming, one of fragment is cut with restriction enzyme, more effective for the method (by restructuring overlapping fragments and carrier in host's body) of the donor gene group being transferred to yeast host cell, this may be due at the more efficient homologous recombination (Orr-Weaver etc. of DNA end, PNAS USA 78,6354 (1981).
Embodiment 1D
Structure carries the diploid yeast bacterium of 2 donor mycoplasma genomes (mycoplasma genitalium and mycoplasma mycoides) Strain
This embodiment has described to produce and has carried 2 genomic diploid yeast host strains of donor mycoplasma, and described 2 donor mycoplasma genomes enter (seeing top embodiment 1A) by the method transfer be depicted in Fig. 2 A.For this process, 2 haploid strains---each carries 1 genome, as at D.C.Amberg etc., Ferment Method in female genetics: cold spring harbor laboratory's procedures manual (Methods in yeast Genetics:A Cold Spring Harbor Laboratory Course Manual) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed.2005,2005), described in pp.230, hybridized (cross).
The W303a bacterial strain that comprises mycoplasma genitalium cI16-2 (mating type a) with VL6-48 bacterial strain (mating type α) mating (seeing top embodiment 1A and table 1) that comprises mycoplasma mycoides LCcI1.1.Before mating, as follows, the HIS3 mark TRP token-replacement in the mycoplasma genitalium genome, to allow selecting to carry two kinds of genomic diploids on the substratum that lacks Histidine and tryptophane.
For with TRP1 displacement HIS3 mark, by PCR from plasmid pRS304 (be described in Sikorski and Hieter, in Genetics 122,19 (1989), Genbank accession number: U03436.1, gi numbering 416305) amplification 1059bp TRP1 fragment.Use has the primer of following sequence, from the fragment of plasmid amplification and mycoplasma genitalium MS5 cIl6-2 homology:
Figure BDA00002358547600771
Figure BDA00002358547600772
CAGAGCAGATTGTACTGAGAGTGCACC (SEQ ID NO:131) and
Figure BDA00002358547600774
ATCTGTGCGGTATTTCACACCGC(SEQ?ID?NO:132)。
In each primer sequence, with the part of mycoplasma genitalium cIl6-2 sequence homology be black matrix.Use Gietz etc., the technology that Nucleic Acids Res 20,1425 (1992) describes, used Lithium Acetate that the 1059bp fragment is transformed into to yeast.
By increasing with 2 primers (sequence: ATTATTCCATCATTAAAAGA (SEQ ID NO:133) and AGTCAAGTCCAGACTCCTGT (SEQ ID NO:134)), confirm that HIS3 is replaced by TRP1, utilize this amplification to produce the 1207bp fragment when HIS3 is replaced by TRP1, produce the 927bp fragment when displacement not occurring.Result shows correct displacement.
After displacement, the haploid strains that carries 2 different donor gene groups is as at D.C.Amberg etc., Yeast Method in genetics: cold spring harbor laboratory's procedures manual (Methods in yeast Genetics:A Cold Spring Harbor Laboratory Course Manual) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed.2005,2005), described in pp.230, hybridized.After hybridization, use the scheme from Bio-Rad CHEF-DR III handbook " cerevisiae dna for preparing the agarose embedding ", in the agarose inserted block from independent clone DNA isolation.The agarose inserted block under constant voltage prerunning several hours to remove yeast chromosomal dna.The DNA separated carries out linearizing in 1 hour by heating under 55 ℃; Control sample does not heat (data do not show).In 5 diploids that result shows in this research to generate whole 5 comprise mycoplasma genitalium and mycoplasma mycoides genome the two, confirm successfully to have generated and comprise 2 genomic diploid yeast host cells of full donor mycoplasma not of the same race.
Embodiment 1E
Retain mycoplasma donor gene group not existing in ARS sequence situation in yeast host cell.
As by Gibson etc., Science 319,1215 (2008) describe and describe the yeast vector in the synthetic mycoplasma genitalium genome generated in embodiment 1C in the above, initial site from it the RNaseP gene is transferred to the novel site in MG411, in order to do not interrupt indispensable gene.For this process, the yeast clone that comprises the synthetic mycoplasma genitalium as described in embodiment 1C in the above and two (2) individual fragments are by cotransformation.The first fragment of long 1842bp, make to comprise URA3, GAL1 promotor and centric yeast vector sequence and insert in MG411.By PCR, use the primer with following sequence to generate this fragment:
Figure BDA00002358547600775
Figure BDA00002358547600776
TTGATTTCGGTTTCTTTGAAAT (SEQ ID NO:135) and
Figure BDA00002358547600777
Figure BDA00002358547600778
(the mycoplasma genitalium sequence is black matrix to GGGTCCTTTTCATCACGTG (SEQ ID NO:136); The NotI site adds Underscore).Template for this PCR is the construct (table 8) with the sequence provided at SEQ ID NO:137.
Table 8. is for the sequence of the pcr template that generates the yeast vector sequence
TTGATTTCGGTTTCTTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCAGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTATCTACTGGAGAATATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTAGAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCCTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATTAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCGGCCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGATCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTTTTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGGACCC(SEQ?ID?NO:137)
Second segment length 302bp has following sequence:
TTAAAGTCAGTTATTTATCTACAGCAATTGCTGTCATTACTTTAATTATTTGTTCTTAAAGCAACATTCCCTTACCAAAATTTAGGTTTCTTGCTTGTGGAGTTTACCGCGTTTCATACCTGGTTTTCACCAAGCTCGTCTCTGTGGCACTTTCAAAACATCATCATAGTATTAACCTTAGACTAGTTATGTCGTTATGGCTATCACATCCTAAATCTTATCGCTTTGATTTACACAAACACTACTTGCATTCCAGCAAGTGCAAGCATGGACTTTCCTCTACTTTAAATATATCTTTAAAG(SEQ?ID?NO:138)。
This fragment is for inserting with mycoplasma genitalium sequence displacement RNaseP yeast vector, in order to recover the coding region of this gene.The primer that utilization has following sequence generates this fragment: TTAAAGTCAGTTATTTATCTACAGC (SEQ ID NO:139) and CTTTAAAGATATATTTAAAGTAGAGG (SEQ ID NO:140) by PCR from mycoplasma genitalium DNA.
Before cotransformation, TRP1 is inserted to MG411, as follows: as to use 2 primers with following sequence, from plasmid pRS304 (Genbank accession number U03436.1, gi number 416305) amplification, there is the 1177bp TRP1 gene fragment with the MG411 homology:
CAGAGCAGATTGTACTGAGA (SEQ ID NO:141) and
Figure BDA00002358547600783
Figure BDA00002358547600784
ATCTGTGCGGTATTTCA(SEQ?ID?NO:142)。In every primer, with the part of mycoplasma genitalium genome homology, with black matrix, list.Pass through PCR, use one group of primer (sequence GCCATTGTTTCACTAATTGC (SEQ ID NO:143) and TAATCCTATCTTTGGAGCTT (SEQ ID NO:144)), confirm that the TRP1 gene inserts, described pcr amplification 1739bp (if insertion) is and if 680bp---not insertion.Be chosen as the cotransformation body of His-Trp-Ura+.By the product of pcr amplification 513bp, use the primer with following sequence, confirm the recovery of RNaseP: CTCCATCATGCGCAGTAATA (SEQ ID NO:145) and CTTTAAAGATATATTTAAAGTAGAGG (SEQ ID NO:146).By the product of pcr amplification 1841bp, use the primer with following sequence, confirm that TRP1 is replaced by yeast vector: TTGATTTCGGTTTCTTTGAA (SEQ ID NO:147) and CAGGCAGGAATTTGATTCCC (SEQ ID NO:148).
The carrier that inserts novel site does not comprise ARS.The result confirmation of these researchs, the carrier that comprises mycoplasma genitalium donor gene group is retained in yeast host cell does not need to exist the ARS sequence.Mycoplasma genitalium is rich in AT and therefore may comprises the sequence that plays ARS effect in yeast.ARS sample sequence is rich in the DNA of AT and often occurs (to see Montiel etc., Nucleic Acids Res 12,1049 (1984) at eucaryon; Stinchcomb etc., PNAS USA 77,4559 (1980)).
Generally speaking, that describes in this embodiment studies confirm that, the method provided is provided, and 3 kinds of different full genomes of donor mycoplasma (maximum size is 1.1MB) is successfully transferred in yeast host cell, and bred in yeast host cell and retain.In each situation, reclaim whole clones and unstable sign do not detected.In addition, in several research, by southern blotting technique, reclaim and detect the molecule that about 2MB is large.These molecules may represent that the clone of concatermer the method that demonstration provides can be used for clone and the larger genome of transfer and nucleic acid molecule and enter yeast host cell.These class methods can be used for generating the host cell that comprises the donor gene group, and the donor gene group then can be bred and modify and be used the method transfer provided to enter recipient cell in host cell.
Embodiment 1F
The genomic stability of mycoplasma mycoides MCpMmyc1.1 and assessment between proliferation period in yeast
Estimate mycoplasma mycoides MCpMmyc1.1 genome stability between proliferation period in yeast.Figure 18 A provides the genomic schematic diagram of YCpMmyc1.1 and shows the position of integrating YCp.Nine (9) the individual independent primers that use in pcr amplification their approximate positions in genome show and number corresponding to the amplicon in Figure 18 B.Detect mycoplasma pneumoniae genome stability between proliferation period in yeast by 2 kinds of methods.In the first, the yeast culture bed board that comprises genomic clone, to upper two day of solid synthetic medium that lacks Histidine, is then pieced together (patch) to new flat board by independent colony.In the second, the yeast culture that comprises genomic clone grows to saturated, is diluted to 1/100 mark and regrows to saturated.Culture was followed bed board to upper two day of solid synthetic medium that lacks Histidine, and then independent colony was pieced together on new flat board.In two kinds of methods, in the multiplex PCR amplification of isolation of genomic DNA 9 independent primer pairs showing, be used as template in using Figure 18 A.Amplicon by the gel electrophoresis analysis gained.Independent primer pair amplicon in the numeral correspondence on gel right side is presented at Figure 18 B.Swimming lane G is positive control and swimming lane N does not have genomic negative control.Molecular weight marker is shown in swimming lane M.The result shown represents 40 analytic samples.All 40 clone's performances comprise the complete genome group, show that bacterial genomes is stable (Figure 18 B) in yeast between conventional proliferation period.
Be evaluated at the mycoplasma mycoides YCP genome operated in III type restriction enzyme allele group in yeast.The genomic schematic diagram of mycoplasma mycoides YCpmycl.l is presented in Figure 18 C; Show the position of integrating YCp.9 independent primer pairs that use in pcr amplification their approximate positions in genome show and corresponding amplicon numbering (the gel result does not show).Diagonal lines in Figure 18 C is illustrated in the amplicon (the gel result does not show) of disappearance in clone 3.After transforming mycoplasma mycoides YCpMmycl.l yeast clone with the box that comprises URA3, assess Ura+ clone's genome by multiplex PCR, and by gel electrophoresis analysis gained amplicon (the gel result does not show).Disappearance amplicon 5 to 8 in clone 3, prompting has large disappearance in this genome.Other 4 clones show and comprise complete genome.
Embodiment 2
The full genome of mycoplasma donor that transfer is bred in yeast host cell enters the mycoplasma recipient cell
Provide for shifting large nucleic acids such as genomic method, described large nucleic acids different biological and cell type (such as, donor, host and acceptor) between shift, it can be not of the same race, boundary and/or purpose (for example different bacterium kinds and bacterium are to the eucaryon yeast cell).Therefore, in some embodiments, the method comprises for overcoming the step of potential uncompatibility between different cell types, such as for successfully the donor gene group of having bred at host cell being transferred to recipient cell.
Following embodiment 2A has described and the method that provides has been provided successfully ((Genbank accession number NZ_AAZK00000000.1 (GI:149364883) is transferred to the recipient cell that difference (mycoplasma capri) is planted to mycoplasma mycoides LC by the full donor gene group of having bred in yeast host cell.This embodiment has described differences the exploitation analyzed between these 3 kinds different biologies (donor, host and acceptors) and can use provided method to be used for overcoming the whole bag of tricks of these differences.
Embodiment 2B shows to reclaim the q.s purifying, complete donor mycoplasma genomic dna from yeast host cell, for being transferred to recipient cell.Embodiment 2C shows that the provided transfer method that enters recipient cell for shifting natural bacteria donor gene group has high-level efficiency.Embodiment 2D has described the restriction modification system (not existing in yeast) in assessment host and recipient cell, and assessment methyltransgerase (it expresses in yeast) is expressed and methyltransgerase on the impact of donor gene group and activation.Embodiment 2E has described the processing used in institute's supplying method with the host that overcomes and restriction-modify (R-M) System Dependent-D-A uncompatibility problem.Embodiment 2F has described the R-M system of mutant receptors cell, and embodiment 2G shows successfully donor gene group (from mycoplasma, transferring to yeast host cell) to be transferred to from yeast host cell different acceptor bacteriums of planting.
Embodiment 2A
Bacterial isolates, culture condition and carrier
For this embodiment and below the research described in embodiment 3, intestinal bacteria DH10B[F --mcrA Δ (mrr-hsdRMS-mcrBC) ф 80dlacZ Δ M15 Δ lacX74deoR recA1 endA1 araD139 Δ (ara, leu) 7697 galU galK λ -RpsL nupG] (Invitrogen, Carlsbad, CA) as the host strain of clone's process and plasmid propagation.Bacillus coli cells is under 37 ℃, at Luria-Bertani (LB) broth culture or grow in LB agar.According to the selective marker existed in given plasmid, the intestinal bacteria transformant is grown in the LB substratum that is supplemented with 50 μ g/ml penbritins, 5 μ g/ml tsiklomitsins or 125 μ g/ml tetracyclines.
This embodiment and below use 2 mycoplasma species kinds in the research described of embodiment 3: mycoplasma capri subspecies capricolum (bacterial strain California Kid TM) (ATCC 27343) and mycoplasma mycoides subspecies Mycoides (bacterial strain GM12) (Damassa etc., 1983; Above-mentioned).The mycoplasma cell, under 37 ℃, is grown (Tully etc. 1977) in the liquid or solid SP4 substratum that comprises 17% foetal calf serum (Invitrogen, Carlsbad, CA).With the mycoplasma of plasmid or complete-genome conversion, under 37 ℃, in the SP4 substratum that is supplemented with 5 μ g/ml tsiklomitsins or 8 μ g/ml tetracyclines, grow.By by mycoplasma bed board detectionofβ-galactosidaseactivity on the solid medium that comprises the chloro-3-indyl-β of the bromo-4-of 150 μ g/ml 5--D-galactopyranoside (X-gal, Promega, Madison, WI).
Below in embodiment 2 kinds of mycoplasma capri subspecies capricolum (mycoplasma capri) bacterial strain as recipient cell: wild-type (wt) mycoplasma capri and the unconfined mycoplasma capri mutant (mycoplasma capri-Δ RE) by the acquisition of inactivation CCATC-restriction enzyme allele in the wild-type mycoplasma capri (below being described in embodiment 2F).Clone (c11.1) for the donor gene group DNA shifted from mycoplasma mycoides subspecies mycoides LC (mycoplasma mycoides LC), be described in top embodiment 1, its genome comprise tetracyclin resistance mark, lacZ gene and be incorporated into ORF04334 (lppA) and ORF04335 (the transposase B of IS1296) between yeast centromeric plasmid.As following detailed description, perhaps, directly from mycoplasma mycoides LC cell clone 1.1 (natural gene group DNA) or describe from embodiment 1 in the above the mycoplasma mycoides that carries generated and clone 1.1 genomic yeast host cells, prepare the mycoplasma genomic dna in the agarose inserted block.
Some carriers that use in embodiment below come from the oriC plasmid, and it can be at mycoplasma mycoides LC (pMYCO1 (SEQ ID NO:149)) with at mycoplasma capri (pMYCOl (SEQ ID NO:149); PSD4 (SEQ ID NO:150)) in, copy (Lartigue etc., Nucleic Acids Res 31,6610 (2003)).Those plasmids are based on pBS (+) plasmid (Stratagene) and comprise the gene of the tetM from transposon Tn916 by the spiralin promoters driven, and as resistance marker, (Lartigue etc., Plasmid 48,149 (2002).The restriction damping fluid is from New England Biolabs, Ipswich, MA.
Embodiment 2B
Separate mycoplasma mycoides LC donor gene group from host's yeast cell, confirm the complete genome DNA reclaimed Quantity and develop transfer method
In order to separate complete mycoplasma donor gene group from yeast cell and to be analyzed, will be with the large colony of mycoplasma mycoides (mycoplasma mycoides LC) GM12, clone the yeast W303 cell that 1.1 genomes transform and be embedded in the agarose inserted block---described in EXAMPLE l A (ii) b and lA (iv) in the above---, as described below.This genome carries tetracycline resistance gene (tetM) and beta-galactosidase gene (lacZ).From inserted block DNA isolation assessment.Do not carry the genomic dna of the unartificial yeast of donor gene group and assess in a similar fashion with the natural donor mycoplasma cell separated, for contrast.
I. the yeast agarose inserted block that comprises the donor gene group
Yeast culture is under 30 ℃, and in selective medium, growth is until OD 600Reach about 1.5.Use is from Bio-Rad Laboratories (Valencia, CA) CHEF mammalian genes group DNA inserted block test kit, according to the scheme of manufacturers's suggestion and following details/improvement, yeast cell is embedded in the agarose inserted block, and from this inserted block DNA isolation.In order to increase the amount of available mycoplasma mycoides LC genomic dna in each inserted block, use every mL inserted block to be prepared 6 * 10 9Individual yeast cell (rather than 6 * 10 8Individual cell), to produce 6 * 10 8The every inserted block of individual cell.After being embedded in inserted block, not to process with lyticase (Bio-Rad Laboratories), but use Zymolyase TM100T enzyme (USB Corporation, Cleveland, OH) digestion is with the digesting yeast cell walls.This enzyme adds the inside or the outside of inserted block to the concentration of 5mg/mL; Mixture is placed 2 hours under 37 ℃.At 1 * TE damping fluid (20mM Tris-HCL pH 8; 50mM EDTA) in, after rinsing, use and Proteinase K reaction buffer (the 100mM EDTA that is supplemented with every ml inserted block 200 μ l Proteinase Ks; 0.2% Sodium desoxycholate; 1% sodium lauroyl sareosine; PH 8.0) at 50 ℃ of lower incubations 2 times, each 24 hours, the yeast cell of cracking embedding (spheroplast) and degrade proteins.The agarose inserted block is then at room temperature at 1 * TE damping fluid (20mM Tris-HCL pH 8; 50mM EDTA) in rinsing 4 this, each 1 hour, follow stirring, and be kept in identical damping fluid under 4 ℃.For the yeast inserted block that will be digested with restriction enzyme (below seeing), the phenylmethylsulfonyl fluoride (PMSF) of adding final concentration 1mM during rinsing for the second time.
Preparation is carried the yeast agarose inserted block of mycoplasma mycoides LC genomic dna and is comprised those that contrast cerevisiae dna, uses from the CHEF mammalian genes group DNA inserted block test kit of Bio-Rad (Valencia, CA) and analyzes.For the yeast (A2, B2 and C2) that comprises the donor gene group with manufacture a series of 3 agarose inserted blocks (A, B and C) for unartificial yeast (Al, Bl and Cl).At room temperature rinsing 2 times 1 hour in 1mL 1 * TE damping fluid of inserted block, and at room temperature 1mL is supplemented with in 1 * NEB damping fluid 2 of BSA (100 μ g/mL) balance 1 hour.
In order to remove endogenous pastoris genomic dna (its in the CHEF gel analysis be described below to mycoplasma mycoides LC genomic dna migration in similar position), inserted block B and C (for every group of yeast) are under 37 ℃, in 500 μ L reaction volumes, with 50 AsiSI of unit, RsrII, together with Fsel restriction enzyme (New England Biolabs), be incubated overnight, described restriction enzyme specificity cutting pastoris genomic dna also keeps donor dna complete.Under identical condition, by inserted block A incubation together with these enzymes.All 3 inserted blocks are at room temperature rinsing 1 hour being loaded onto in 1%TAE sepharose (120 minutes, 120 volts), to shift out the pastoris genomic dna fragment of digestion from inserted block in 1 * TE damping fluid then.
After migration, the agarose inserted block is removed and rinsing twice 1 hour in 1mL 0.1 * TE damping fluid from well, and in 1mL is supplemented with 1 * NEB damping fluid 2 (New England Biolabs, Ipswich, MA) of BSA (100 μ g/mL) balance 1 hour.For linearizing mycoplasma mycoides LC genomic dna, allow DNA to enter gel, inserted block C under 37 ℃ together with 50 PspXI of unit restriction enzymes incubation.Inserted block A and B (for every group of yeast) not enzyme-added incubation under identical condition is used for simulation-digestion (mock-digestion).After incubation, all inserted blocks are at room temperature used 1mL 1 * TE damping fluid rinsing 1 hour, and application of sample is to pulsed field gel.
Ii. natural mycoplasma mycoides LC agarose inserted block
In order relatively to reclaim the amount of DNA, use is from Bio-Rad (Valencia, CA) CHEF mammalian genes group DNA inserted block test kit, the natural donor mycoplasma mycoides LC agarose inserted block of the genomic dna that also preparation comprises different amounts from the mycoplasma mycoides cell.As Lartigue etc., Science 317,632 (2007) is described---have some improve, particularly separate before the mode of cell cultures, carry out separating complete complete genome DNA from mycoplasma mycoides LC.500 (500) mL mycoplasma mycoides LC (tetM, lacZ, YCp) cells are grown in the SP4 substratum that is supplemented with 10 μ g/ μ l tsiklomitsins and l0 μ g/ μ l Streptomycin sulphate, until the pH of substratum reaches 6.5 (about 10 9Individual cell/mL).Before collecting cell, add 100 μ g/ μ l paraxin to substratum, and, at 37 ℃, under this cell concn, the incubation cell is other 90 minutes, in order to make ongoing chromosome duplication wheel subsynchronous, and suppresses copying of next round.
In 10mM Tris pH 6.5,0.5M sucrose, the rinsing cell once, and is resuspended in the damping fluid that 2mL is identical.From this cell suspending liquid, by 2 times of serial dilution mycoplasma mycoides LC cells, prepare 8 series (progression, seires) mycoplasma mycoides LC genomic dna (MLC gDNA) agarose inserted block.The every inserted block of inserted block from series 1 approximately comprises 10 10Individual natural mycoplasma mycoides LC cell; The every inserted block of inserted block from series 7 approximately comprises 1.5x10 8Individual; Approximately comprise 7x10 with the every inserted block of inserted block from series 8 7Individual.In order to compare the genomic dna from yeast, by PspXI the digestion inserted block that comprises n DNA and the inserted block (see above) that comprises the mycoplasma mycoides LC genomic dna separated from yeast, so that the linearizing of MLC genome, as mentioned above, for analyzing on pulsed field gel.
Iii. the genomic dna relatively reclaimed on pulsed field gel
By many amounts of the amount that compares the genomic dna separated in the agarose inserted block from yeast cell and the natural gene group DNA separated, estimate the amount of mycoplasma mycoides LC genomic dna in yeast cell from the mycoplasma mycoides LC cell of 2 times of serial dilutions in the agarose inserted block.For this process, yeast agarose inserted block (embodiment 2B (i), inserted block Al, Bl and Cl and A2, B2 and C2) and eight mycoplasma mycoides LC agarose inserted blocks (embodiment 2B (ii)), 1% pulsed field sepharose (Bio-Rad verified in TAE 1X, Valencia, CA) in, with clamper uniform electric field (Chu etc., Science 234,1582 (1986)) (CHEF DR III; Bio-Rad) carry out electrophoresis.Burst length slow is raised to 120 seconds from 60, under 4.5V/cm 27 hours.After migration, use
Figure BDA00002358547600831
GOLD nucleic acid staining agent (Invitrogen, Carlsbad, CA) (1/10,000 dilution) dyeing gel, and GE Typhoon 9410 imager scannings for the PFGE pattern.The large tick marks of yeast saccharomyces cerevisiae CHEF DNA is for assessment of the DNA size; This mark comprises yeast saccharomyces cerevisiae karyomit(e) and the size in the 0.2-2.2Mb scope for amount.
Swimming lane with the inserted block A2-C2 yeast of self-contained mycoplasma mycoides donor gene group (come) and A1-C1 (unartificial yeast) (embodiment 2B (i)) together with the mark swimming lane on gel electrophoresis.Inserted block (organizing 7 and 8) comprises increases the mycoplasma mycoides of concentration n DNA (embodiment 2B (ii)).In some swimming lane, the desired location place of pulsed field gel detects 1.12Mb mycoplasma mycoides LC genome.In the yeast host cell that comprises mycoplasma mycoides donor gene group, select the digesting yeast genomic dna with enzyme mixture (AMSI, RsrII, FseI restriction enzyme), then electrophoresis (sample B2 and C2; See top embodiment 2B (i)) recovery (data do not show) that improved mycoplasma mycoides donor gene group DNA.In addition, linearizing mycoplasma mycoides LC genome (C2) has improved the recovery of 1.2Mb mycoplasma mycoides (CT) greatly.In the parallel sample of the yeast cell that does not comprise mycoplasma mycoides genome (B1, C1) from natural (wild-type), 1.2Mb band do not detected, confirm that band at 1.2Mb represents really to have the mycoplasma mycoides genome in cell in yeast.Identical band appears in the swimming lane that comprises natural mycoplasma mycoides genomic dna.
The amount of the mycoplasma mycoides LC genomic dna reclaimed from yeast cell is with from natural mycoplasma mycoides LC genomic dna standard substance, the amount of---its also as above-mentioned process with PspXI---compares.From 6 * 10 8The amount of the mycoplasma mycoides LC genomic dna of individual yeast cell acquisition and natural mycoplasma mycoides LC inserted block (the every inserted block about 1.5 * 10 from from series 7 8The amount of the genomic dna individual natural mycoplasma mycoides LC cell) reclaimed is similar.
Iv. quantize the DNA reclaimed
The UV spectrophotometer is for measuring the amount of the natural mycoplasma mycoides LC genomic dna existed at the melting inserted block prepared from natural mycoplasma mycoides cell, as described in embodiment 2B (ii).Result is listed in the following Table 9.As shown in this table, the inserted block from serial 7 comprises about 12ng/ μ l mycoplasma mycoides LC genomic dna (every 100 μ L inserted block 1.2 μ g).As above recording, pulsed field gel shows suitable 1.2Mb band strength the swimming lane with this sample (7) and the swimming lane that comprises the DNA reclaimed from yeast samples (C2) (embodiment 2B (iii) above seeing).Therefore, in definite every 100 μ L inserted blocks that reclaim from the host cell that comprises the donor gene group, the amount of mycoplasma mycoides LC genomic dna is substantially equal to 1 μ g.
Table 9: quantification and the transfer of natural mycoplasma mycoides LC genomic dna
Figure BDA00002358547600851
Embodiment 2C
The DNA that transfer is reclaimed from inserted block enters recipient cell
For natural mycoplasma mycoides LC agarose inserted block (the embodiment 2B (iv) from melting; Table 9) each sample of series, the inserted block by 1/5 (20 μ L) shifts and enters the mycoplasma capri recipient cell, its use and Lartigue etc., the similar scheme that Science 317,632 (2007) describes, have some improvement, as follows:
I. culture and prepare recipient cell
Six (6) mL mycoplasma capri recipient cells are being supplemented with foetal calf serum (17%), glucose (10g/L), 2ml phenolsulfonphthalein (1%) and 100 μ l penicillin Gs (5mg/ml)) SOB (+) substratum (Bacto SOB substratum (Becton Dickinson; Franklin Lakes, NJ)) middle growth, until the pH of culture reaches pH 5.7 to 5.85 (about 5 * 10 7Individual cell/ml).Recipient cell under 10 ℃, with 4575g centrifugal 15 minutes, (10mM Tris-HCl, pH 6.5 in the S/T damping fluid; With 250mM NaCl) rinsing once, be resuspended in 200 μ lCaCl 2(0.1M) in, and incubation on ice 30 minutes.
Ii. the donor gene group DNA that preparation separates in the agarose inserted block
Before transfer, the agarose inserted block that comprises mycoplasma mycoides LC genomic dna (serial 1-7) is the stirring of gentleness at room temperature, and in 0.1 * TE damping fluid [2mM TRIS-HCl, pH 8.0,5mM EDTA], rinsing is 2 times, each 30 minutes.Remove damping fluid fully and under 65 ℃, with 10 * β of 1/10 volume-gelase damping fluid [10mM Bis Tris-HCl pH 6.5; 1mM Na 2EDTA] melt agarose inserted block 10 minutes.The agarose melted is cooled to 42 ℃, continue 10 minutes, and, at this temperature, the β of every 100 μ l inserted blocks and 3 units-gelase I (New England Biolabs, Ipswich, MA) is incubated overnight together.
Iii. use 5%PEG to shift
On ice after 30 minutes, 200 μ l recipient cells comprise with 400 μ l the agarose inserted block (1/5 inserted block) that 20 μ l melt donor gene group DNA---as generated in embodiment 2B (ii) in the above---SP4 (-) substratum leniently mix.As following, just, before carrying out next step, add inserted block.
2 of equal-volume (620 μ l) * fusion damping fluid (20mM Tris-HCl pH 6.5,250mM NaCl, 20mMMgCl 2, 10% Fluka PEG-6000 (Sigma-Aldrich, St.Louis, MO)) add to immediately in the mixture of SP4 (-), genomic dna and cell, and by leniently shake the pipe within 30 seconds, make mixture even.Under 37 ℃, after 50 minutes, add the SP4 of 5ml pre-warm cell mixing leniently.Under 37 ℃, after another 3 hours, under 10 ℃, with 4,575g eccentric cell 15 minutes, be resuspended in 0.6ml SP4 and bed board on the SP4 flat board that comprises 4 μ g/ml tsiklomitsins and 150 μ g/ml X-gal.After 3-4 days, select independent colony, and grow in the broth culture that comprises 10 μ g/ml tsiklomitsins.
Result provides in table 9 in the above, and it shows that the amount of the natural mycoplasma mycoides LC genomic dna that the quantity of the transformant (the recipient cell colony with donor dna) of recovery exists relatively in shift reaction is increase pro rata.The quantity of observing transformant under the high DNA concentration (series 8) detected reduces.Obtain 30 (30) individual transformant with the 200ng genomic dna.In the experiment of using subsequently the method, the natural mycoplasma mycoides LC of every 1 μ g donor gene group DNA obtains about 200 transformant colonies usually.These data show that the efficiency of the party's case is sufficiently high, and the quantity of the donor mycoplasma mycoides LC DNA obtained from yeast host cell is not restrictive factor in the genome that uses the method shifts.
Replace with 10 μ g plasmid DNA by the agarose inserted block in solution, 20 μ L melted, above-mentioned transfer method also can be used for transforming the mycoplasma capri recipient cell by plasmid DNA (not in the agarose inserted block).
Embodiment 2D
Assessment restriction-modification (R-M) system
Because between donorcells, host cell and recipient cell, the difference of restriction modification system may cause genome to shift difficulty, so study these systems.This embodiment illustrated in some mycoplasma cells that use in this paper shifts and existed and the component of the restriction modification system of activation, and some aspects that the method provided has been described are for destroying (subvert) these systems successfully to shift.
I. identify the prediction R-M system in donor and recipient cell
Mycoplasma mycoides LC genome (the Genbank accession number: NZ_AAZK00000000 that prediction has been sequenced; GI:149364883) comprise 6 different restriction modification systems (5 II type systems and 1 III type system).The mycoplasma capri genome sequence shows 1 the II type system that exists.From this gene order (Dr.R.Roberts, private letter) prediction II type enzyme recognition site specificity, as at Roberts RJ etc., Nucleic Acids Res.35 (database periodical): biology described in D269-70 (2007).Also referring to REBASE, the restriction enzyme database, can be at Web address: rebase.neb.com/rebase/rebase.html obtains.These recognition sites and the commercial available restriction enzyme in these site cuttings are listed in the following Table 10.The specificity of III type system is prediction not.
Table 10: from the restriction of mycoplasma capri and mycoplasma mycoides LC sequence prediction-modification (R-M) system
Figure BDA00002358547600871
The confirmation of ii.R-M system
A. the methylation state of restriction site
Use the restriction enzyme isoschizomer of the commercial available II type restriction enzyme system corresponding to prediction to confirm that the prediction site of natural gene group in being listed in table 10 of mycoplasma mycoides LC and mycoplasma capri methylated.With isoschizomer digestion mycoplasma mycoides LC and the indication of mycoplasma capri genomic dna, natural gene group DNA is methylated (data do not show) in the site of prediction.The two comprises the CCATC restriction modification system these results demonstration mycoplasma capris and mycoplasma mycoides LC.
For this research, use
Figure BDA00002358547600872
Genomic dna purification kit (Promega, Madison, WI), according to the specification sheets of manufacturers, purifying is from the genomic dna of mycoplasma mycoides LC and mycoplasma capri.As manufacturers (New England Biolabs, Ipswich, MA) is described, by approximately separately the mycoplasma mycoides LC of 1 μ g and mycoplasma capri genomic dna respectively with BccI, HinfI, HpyAV, MboI and SfaNI incubation.Then by agarose gel electrophoresis analyzing DNA (data do not show).
As desired as the R-M system (in Table 10) based on prediction, the cutting of 5 kinds of restriction enzyme isoschizomers of the anti-all tests of mycoplasma mycoides LC genomic dna, show that DNA is methylated in these each site.On the other hand, the mycoplasma capri genomic dna is the cutting of anti-restriction enzyme isoschizomer (BccI) only, and this restriction modification system is all identified in two biologies.These results verifications these separately R-M system (table 10) in the mycoplasma kind, exist, and display case as mycoplasma mycoides LC and mycoplasma capri, the two all comprises the CCATC restriction modification system.
Whether corresponding to the availability of the commercial restriction enzyme isoschizomer of the restriction enzyme system from mycoplasma capri prediction, make us can test 2 genomes is methylated at suitable restriction site place.Use the genomic dna of Wizard genomic dna purification kit purifying from mycoplasma mycoides and mycoplasma capri.By mycoplasma mycoides and mycoplasma capri genome and the BccI incubation of about 1 μ g separately.Then by the agarose gel electrophoresis analyzing DNA.As expected, the two all cutting of anti-BccI of mycoplasma mycoides and mycoplasma capri genomic dna, described BccI is the enzyme (data do not show) corresponding to the homology R-M system of 2 biologies.
B. restriction enzyme activity
Prepare cell-free extract from mycoplasma mycoides LC and mycoplasma capri, to show that restriction enzyme activates in two kinds.As following, described, extract is used in the restriction enzyme test, to test them, cuts the ability of mycoplasma mycoides LC, mycoplasma capri and mycoplasma genitalium genomic dna.
C. prepare cell extract
For mycoplasma mycoides LC, 1 liter of cell culture in the SP4 substratum is grown under 37 ℃, until reach the pH of 6.2-6.3.Part by cell being divided into to 5 200mL under 4 ℃ in the SLA-1500Sorvall rotor with 5,000 * g centrifugal 15 minutes, the results culture.Each mycoplasma mycoides LC throw out is then with the Hepes of 200mL 8mM, the sucrose rinsing of pH 7.4 and 272mM, under 4 ℃ in the SLA-1500Sorvall rotor with 5,000 * g centrifugal 15 minutes.Each gained throw out is resuspended in (20mM Tris-HCl, pH 7.5,0.1mM EDTA, 150mM NaCl, 1mM DTT and 10% glycerine) in the 1ml Extraction buffer, and use subsequently output to control probe (microtip) that (output control) is 3 in supersound process on ice 5 times, 10-12 pulse per second (PPS) (brust).By under 4 ℃, with 18,000 * g microcentrifugation 30 minutes, make each solution clarification.Combine the soluble part of each gained, test proteins concentration, be divided into 200 μ l parts, and preserve under-80 ℃.The protein concentration scope of the extract prepared in this mode is typically from 15 to 25mg/ml.
For mycoplasma capri, except 1 liter of culture is grown in the SP4 substratum that comprises 10 μ g/ml tsiklomitsins, use with the same procedure that mycoplasma mycoides LC is described and prepare extract.
D. restriction enzyme activity identification
Active following mensuration of the restriction enzyme of mycoplasma mycoides LC extract.Use
Figure BDA00002358547600881
Genomic dna purification kit (Promega Corporation, Madison, WI) individually in each autoreaction, two (2) the μ g genomic dnas that separate with mycoplasma genitalium from mycoplasma capri, mycoplasma mycoides LC, under 37 ℃, incubation in 1 * NEB restriction enzyme damping fluid 4 of cumulative volume 100 μ l adds 100 μ M deoxynucleotides and 8 μ g extracts.Finally, with 0,5,10 and 15 minutes intervals, add albumen, shift out 20 μ l aliquots containigs, and add in 20 μ l 2 * stop buffers (2% SDS, 20mM EDTA).Extract solution at room temperature with 18,000 * g centrifugal 2 minutes with 40 μ l phenol/chloroform/primary isoamyl alcohol (25:24:1).Water is put into has 10 of 4 μ l * blue juice (Blue Juice) (Invitrogen, Carlsbad, CA) in new pipe, and each solution to 0.8%1 of application of sample 18 μ l * TAE sepharose electrophoresis 16 hours under the FIGE condition of 120V, 0.1-0.6 linearity and 80V, 0.1-0.6 linearity.Sepharose is then used
Figure BDA00002358547600882
GOLD nucleic acid staining agent (Invitrogen, Carlsbad, CA) (1/10,000 dilution) is dyeed 30 minutes, and scans with GE Typhoon 9410 imagers.
Except in each reaction, using 1 * NEB restriction enzyme damping fluid 1 and 12 μ g mycoplasma capri extracts, detect the restriction enzyme activity of mycoplasma capri extract with identical method, and aliquots containig shifts out and processes with 0,15,30 and 45 minute interval.
The result of study (the gel result does not show) of describing in (a) at embodiment 2D (ii) shows; mycoplasma mycoides LC genomic dna should protectedly be avoided the restriction modification system cutting of mycoplasma capri, but the mycoplasma capri genomic dna should easily be cut by mycoplasma mycoides LC restriction modification system.In fact, as predicted by the homology restriction modification system, from the genomic dna of mycoplasma mycoides LC and cut from the genomic dna of mycoplasma capri, and it is easily cut not comprise the genomic dna from mycoplasma genitalium of any restriction modification system.Cutting mycoplasma genitalium genomic dna is the activity due to restriction enzyme in mycoplasma capri.This is proved by following truth: the crude extract that comes from the mycoplasma capri bacterial strain---wherein prediction restriction enzyme allele destroyed (seeing embodiment 2F)---, does not cut any the genomic dna from 3 kinds of mycoplasma bacterial strains that detect.Also as expected, when incubation together with mycoplasma mycoides LC crude extract, cut and not cut from the genomic dna of mycoplasma mycoides LC from the genomic dna of mycoplasma capri.These results show that mycoplasma capri and mycoplasma mycoides LC comprise active restriction modification system, and it has the potential of impact from the activity of the unmethylated mycoplasma mycoides LC donor gene group of yeast separation.λ DNA is digested by wild-type mycoplasma capri extract.By DNA incubation together with the extract of manufacturing from mycoplasma capri RE (-) bacterial strain, do not cause indicating the non-existent band of this bacterial strain restricted activity to occur.
E. mycoplasma mycoides donor and transfer body clone's gene order-checking and genome are relatively
Two mycoplasma mycoides clones are sequenced.One is the donor gene group, it is by Lartigue etc., in 2007 at Science (317,362) disclosed " genome in bacterium shifts: make a kind of species become another kind (Genome transplantation in bacteria:changing one species to another) " article describes (1,088,905bp, GenBank# CP001621).Another is to comprise III type restriction enzyme allele disappearance (Δ typeIIIres; The mycoplasma mycoides clone (l, 084,586bp, Genbank # CP001668) of transfer Figure 17).The clone who uses in the article of 2007 only uses Sanger DNA sequencing chemistry to be checked order.III type restriction enzyme allele deletion clone described herein checks order and covers and also use 454FLX to become opposite end to read tetra-sodium order-checking chemistry (paired-end read pyrosequencing chemistry) order-checking to 8x by the Sanger method.
In order to confirm that transformant is all mycoplasma mycoides rather than comprises the yeast sequence or the mosaic of mycoplasma capri recipient cell sequence, and, in order to confirm when being cloned into yeast, whether bacterial genomes is stable, relatively 2 sequences.The Δ typeIIIres genome coupling mycoplasma mycoides of all assemblings, except those zones of coupling YCp carrier.In addition, except the difference of deliberately manufacturing in genome, the transformant mycoplasma mycoides genome sequence with III type restriction enzyme allele disappearance is identical with the mycoplasma mycoides genome checked order before, except site 95.It should be noted that between the genome of donor mycoplasma mycoides bacterial strain that these 95 sequence differences are used in this article and transformation and there is no difference.Between the genome (Genbank # CP001668) of the mycoplasma mycoides bacterial strain sequence (Genbank #CP001621) in 2007 articles (Id.) and this paper transformation, they are difference.Our order-checking is each of---it is for generating initial mycoplasma mycoides yeast clone---those 95 sites at mycoplasma mycoides YCpMmycl.l.In each example, shift its YCpMmycl.l donor come from of body sequences match.Therefore, clone in yeast, breed in yeast and transform or migrate out yeast with during manufacturing the mycoplasma mycoides bacterial strain of transforming, not causing any 95 sequence differences.Hypothesis based on such: all sequences identical in 2 mycoplasma mycoides genomes that check order fully is also identical in the YCpMmycl.l F+strain, reach a conclusion, except those of intentional transformation, in yeast clone and propagation and be transferred back to bacterium during do not have sequence to change.
These data show the not restructuring of yeast or recipient cell genome and mycoplasma mycoides donor gene group, and these bacterial genomes sequences in yeast as being stable between YCp propagation, transformation and preservation period.
Embodiment 2E
Avoid the incompatible sex method of restriction modification system for the protection of donor gene group and nucleic acid
This embodiment has described two kinds of methylation methods for example, avoiding the cutting of be limited-modification system for the protection of mycoplasma (, mycoplasma mycoides LC) donor gene group.Every kind of method is methylated to test to confirm effect.
I. the methyltransgerase by structure and purifying methylates
At first, as following, described, definite genomic sequence of mycoplasma mycoides LC is for the methyltransgerase (seeing top table 10) of heterogenous expression each evaluation of purifying.Because unique R-M system of identifying in mycoplasma capri with in mycoplasma mycoides LC, identify identical, so only have mycoplasma mycoides LC methyltransgerase to be purified for this research.
A. methyltransgerase builds
From the encoding sequence of the methyltransgerase (CCATC-M, CCTTC-M, TypeIII-M, GCATC-M and GANTC-M) of 5 evaluations of the potential restriction modification system in mycoplasma mycoides LC by codon optimized, for expressing at yeast.Then by 5 ' exonuclease, archaeal dna polymerase and DNA ligase teamwork, use a step isothermal dna assembly method to build these sequences from many overlapping 60bp oligonucleotide, described assembly method is described in Gibson etc., Nature Methods 6,343-345 (2009) and in the Application No. 12/371,543 of submitting on February 19th, 2009.Briefly, utilize the method, at first DNA fragmentation caves in (recess) by 5 ' exonuclease, produces the strand overhang, and it is annealing specifically then, then uses polysaccharase and ligase enzyme fill up the gap covalently bound.After structure, CCATC-M, CCTTC-M and TypeIII-M sequence are cloned into pTYBl expression vector (New England Biolabs, Ipswich, MA; SEQ ID NO:156).GCATC-M and GANTC-M sequence are used
Figure BDA00002358547600901
Recombinant clone technology (Invitrogen, Carlsbad, CA) is cloned into the histidine-tagged expression vector of N-end.
B. methylase purifying
CCATC-M, CCTTC-M expression plasmid
CCATC-M and CCTTC-M expression plasmid are transformed into the positive cell (Stratagene of BL21 (DE3) codon, La Jolla, CA), and transformant is used to inoculate individually the ZYM-505 substratum (Studier that 250ml comprises 100mg/ml Pyocianil and 34mg/ml paraxin, FW, Protein Expr Purific41:207-34 (2005)), powerful vibration (315rpm) growth and under 37 ℃.After about 4 hours, culture is transferred to 16 ℃ also by adding 0.3mM IPTG abduction delivering, spend the night.Cell is agglomerating, be suspended in 50mlIntein lysis buffer (25mM HEPES-NaOH pH 7.2,500mM NaCl, 1mM EDTA, 10% glycerine, positive proteinase inhibitor (plus protease inhibitor) (adequate proteins enzyme inhibitors mixture, Roche Applied Sciences, Indianapolis, IN)) in, and pass the high-pressure homogenizer cracking by twice.
Lysate (lysate) is by under 4 ℃, with 20,000 * g clarification in centrifugal 20 minutes.The clarification lysate is in the post of 1.5ml chitin pearl, by the suggestion purifying of manufacturers (New England Biolabs, Ipswich, MA).Concentrate the part that comprises suitable methyltransgerase and enzyme buffer liquid (50mM HEPES-NaOH pH7.2,50mM NaCl, 0.1mM EDTA, 10% glycerine) is dialysed.After dialysis, use the concentrated methyltransgerase of Amicon ultracentrifugation filter for installation (Millipore, Billerica, MA).
TypeIII-M
Use identical scheme purifying TypeIII-M albumen, after purifying on the post on the chitin pearl, use HiTrap MonoQ post (GE Heathcare) to be further purified albumen.Protein is written into to buffer A (50mM HEPES-NaOH pH 7.2,50mM NaCl, 1mM EDTA, 10% glycerine), and uses the linear gradient elution of from 0 to 100% buffer B (50mM HEPES-NaOH pH 7.2,1M NaCl, 1mM EDTA, 10% glycerine).Concentrate the part that comprises TypeIII-M, dialysis enters the enzyme buffer liquid that comprises 100mM NaCl, and, as CCATC-M and CCTTC-M protein are described, is concentrated.
GCATC-M and GANTC-M expression plasmid
M.GCATC and M.GANTC expression plasmid are transformed into to the positive cell of BL21 (DE3) codon and transformant for inoculating individually the ZYM-505 substratum that 2ml comprises the 100mg/ml Pyocianil, and under 37 ℃ powerful vibration overnight growth.1 milliliter of culture spent the night is for inoculating 250ml ZYM-5052 substratum (Studier, FW, Protein Expr Purific 41:207-34 (2005)).Cell is powerful the vibration 20 hours under 27 ℃ then.Cell is agglomerating, be suspended in 50ml Nickel lysis buffer (50mM HEPES-NaOH pH7.2,500mM NaCl, 30mM imidazoles, 10% glycerine, positive proteinase inhibitor (plus protease inhibitor) (adequate proteins enzyme inhibitors mixture, Roche Applied Sciences, Indianapolis, IN)) in, and pass the high-pressure homogenizer cracking by twice.Lysate is by under 4 ℃, with 20,000 * g clarification in centrifugal 20 minutes.The clarification lysate is used 5ml HisTrap column purification, and the Nickel lysis buffer is as electrophoretic buffer and have the Nickel lysis buffer of 300mM imidazoles as elution buffer.Concentrate M.GCATC albumen, dialyse into the enzyme buffer liquid that comprises 100mM NaCl, and as above concentrated.The utilization of M.GANTC albumen is further used 1ml HiTrap MonoQ heparin column purifying as the buffer A for M.TypeIII and B.Concentrate the part that comprises M.GANTC, dialyse into the enzyme buffer liquid that comprises 100mM NaCl, and as above-mentioned concentrated.
C. methyltransgerase research
The methyltransgerase of purifying is used the plasmid to determine whether they can methylate and comprise mycoplasma DNA in the test that methylates.Use Wilson and Hoffman, the buffer conditions that Anal Biochem 191,370 (Dec, 1990) the describes test that methylated.Under 37 ℃, in 100 μ l volumes, reacted.Reaction mixture comprises 100mM Tris-HCl, pH 7.5,10mM EDTA, 3 μ M DTT, 200 μ M S-adenosylmethionines (SAM), 3 μ g pSmart-pMYCOl plasmid DNA (yeast-intestinal bacteria-mycoplasma three shuttle vectorss).
Methylate in order to assess the methyltransgerase whether DNA be purified, use the restriction enzyme isoschizomer of buying from New England Biolabs, cut each sample of 4 μ l according to the specification sheets of manufacturers.The restriction enzyme isoschizomer used depend on evaluated methylated sequence (BccI (and recognition site, CCATC), HinfI (GANTC), HpyAV (CCTTC), SfaNI (GCATC).Sample was upper at 1% 48-hole agarose E-gel (Invitrogen, Carlsbad, CA), with 70V electrophoresis 25 minutes.Gel scans in GE Typhoon 9410 imagers.
Result shows independent purifying Methyl transporters endonuclease capable Economical Purification mycoplasma plasmid DNA, as corresponding restriction enzyme isoschizomer after the methyltransgerase by with independent (in Table 10) and S-adenosylmethionine (SAM) incubation can not cut plasmid DNA (gels data does not show) judgement fully.
In another research, in the situation that be with or without SAM, use the mycoplasma mycoides crude extract to process the pMYCOl plasmid DNA.DNA is then digested to check the activity (data do not show) of methyltransgerase in extract by BccI.The extract of mycoplasma mycoides is also protected the donor dna entered.Other restriction modification systems that exist in mycoplasma mycoides donor gene group do not affect transfer.
Ii. by crude extract, methylate
To be carried out in order eliminating the possibility (methylase therefore be not purified solves) that in comfortable genome sequence, the enzyme of not identifying restriction modification system of evaluation does not even cut after methylating, to have been developed the scheme of using the crude extract methylate DNA prepared from mycoplasma capri and mycoplasma mycoides LC.Suppress the EDTA of nuclease by adding 10mM, extract can be used as the source of methyl transferase activity.
A. prepare cell extract
For mycoplasma mycoides LC, 1 liter of cell culture in the SP4 substratum is grown under 37 ℃, until reach the pH of 6.2-6.3.Part by cell being divided into to 5 * 200mL under 4 ℃ in the SLA-1500Sorvall rotor with 5,000 * g centrifugal 15 minutes, the results culture.Each mycoplasma mycoides LC particulate matter is then with the Hepes of 200mL 8mM, the sucrose rinsing of pH 7.4 and 272mM, under 4 ℃ in the SLA-1500Sorvall rotor with 5,000 * g centrifugal 15 minutes.Each gained throw out is resuspended in (20mM Tris-HCl, pH 7.5,0.1mM EDTA, 150mM NaCl, 1mM DTT and 10% glycerine) in the 1ml Extraction buffer, and use subsequently output to control to be 3 probe in supersound process on ice 5 times, 10-12 pulse per second (PPS).By under 4 ℃, with 18,000 * g centrifugal 30 minutes, make each solution clarification.Combine the soluble part that each obtains, test proteins concentration, be divided into 200 μ l parts and preserve under-80 ℃.The protein concentration scope of the extract prepared in this mode is typically from 15 to 25mg/ml.
For mycoplasma capri, except 1 liter of culture is grown in the SP4 substratum that comprises 10 μ g/ml tsiklomitsins, use with the same procedure that mycoplasma mycoides LC is described and prepare extract.
B. by the crude extract evaluation, methylate
The use test that methylates shows the methylate ability of the mycoplasma plasmid DNA of growing in various host cells of crude extract, as follows.Use Wilson and Hoffman, the buffer conditions that Anal Biochem 191,370 (1990) describes is used the test that methylates of crude extract.
C. assess mycoplasma mycoides LC extract methylating to CCATC, GANTC, CCTTC and GCATC
In order to assess methylate (and the therefore protection) to CCATC, GANTC, CCTTC and GCATC site of mycoplasma mycoides LC extract, under 37 ℃, in 100 μ l volumes, reacted.Reaction mixture comprises 100mM Tris-HCl, pH 7.5,10mM EDTA, 3 μ M DTT, 200 μ M S-adenosylmethionines (SAM) (do not exist in control sample, as noted), and 20 μ g mycoplasma mycoides LC extracts the pMYCOl plasmid DNA that 3 μ g separate from intestinal bacteria (SEQ ID NO:149)).
With 0,2,4 and 16 hours intervals, shift out 20 μ l aliquots containigs and be added in 2 * stop buffer (2% SDS, 20mM EDTA) from each reaction mixture.To each aliquots containig, use 40 μ l phenol/chloroform/primary isoamyl alcohol (25:24:1) to carry out DNA extraction, then at room temperature, with 18,000 * g centrifugal 2 minutes.Each water is put into new pipe, and by adding ice-cold 100% ethanol of 80 μ l and 1 μ l
Figure BDA00002358547600931
GlycoBlue TM(Invitrogen, Carlsbad, CA) also is incubated overnight under-20 ℃, precipitation DNA.Each sample then under 4 ℃ with centrifugal 15 minutes of 18,000 * g and with 100 μ l ice-cold each gained throw out of 70% ethanol rinsing.With after centrifugal 15 minutes of 18,000 * g, abandon each supernatant liquor under 4 ℃, and dry each throw out, and be resuspended in 20 μ l TE pH 8.0.
In order to assess DNA, whether by II type methyltransgerase separately, methylated, use and buy from New England Biolabs, the restriction enzyme isoschizomer that is applicable to evaluated particular sequence, explanation according to manufacturers, cut 4 each sample of μ l (BccI (and recognition site, CCATC), HinfI (GANTC), HpyAV (CCTTC), SfaNI (GCATC).Sample was upper at 1% 48-hole agarose E-gel (Invitrogen, Carlsbad, CA), with 70V electrophoresis 25 minutes.Gel scans in GE Typhoon 9410 imagers.
D. assess the mycoplasma mycoides LC extract GATC site that methylates
When test mycoplasma mycoides LC extract methylates to GATC, the pMYCOl plasmid DNA of separating from mycoplasma capri by the 3 μ g that use in first step and with the cutting of MboI restriction isoschizomer, improve top scheme.
E. assess the mycoplasma capri LC extract CCATC site that methylates
As above test by the mycoplasma capri extract CCATC site that methylates, except each of the pSmart-pMYCOl plasmid DNA (yeast-intestinal bacteria-mycoplasma three shuttle vectorss and the pMYCOl separated with mycoplasma mycoides LC from mycoplasma capri) of separating from intestinal bacteria of using 3 μ g, and exist or do not exist in situation at S-adenosylmethionine and carry out reacting in 4 hours.Then by with the BccI incubation, check methylated DNA, as above.
F. result
2 plasmids that the mycoplasma capri crude extract can exhaustive methylation separates from intestinal bacteria, it is proved by following truth: corresponding restriction enzyme isoschizomer BccI, can not cut the plasmid (gels data does not show) with crude extract and SAM incubation.
The result of the research experiment in (and therefore protection) CCATC, GANTC, CCTTC, GCATC and GATC site is as follows for methylating by mycoplasma mycoides LC extract: in the situation that 4 restriction modification systems; mycoplasma mycoides LC extract can exhaustive methylation mycoplasma plasmid DNA, and in the situation that 1 system (GATC restriction modification system) part methyl (the gel result does not show).Because endogenous mycoplasma mycoides LC GATC methyltransgerase shows low activity in mycoplasma mycoides LC extract, commercial available intestinal bacteria dam methyltransgerase (New England Biolabs, Ipswich, MA) for successfully supplementing its activity.
Significantly improved the transformation efficiency (data do not show) in mycoplasma capri by two kinds of crude extracts mycoplasma plasmid DNA that methylates, this shows can help to overcome the potential uncompatibility that will be transferred the donor gene group that enters the mycoplasma capri acceptor of breeding with the crude extract incubation in yeast cell in provided method.The effect of III type methyltransgerase is not assessment in mycoplasma mycoides LC crude extract, because its locus specificity the unknown.
Iii. with the increase transformation efficiency that methylates
Unmethylated plasmid and described above with the methylated plasmid of mycoplasma mycoides LC extract, be transformed into the mycoplasma capri cell and compare.Result indicates methylated plasmid to increase transformation efficiency, the positively effect of DNA modification before showing to transform.
Iv. the processing of other analysis, conformation effect and DNA after methylating
Although crude extract protection donor plasmid DNA avoids the impact of host's restriction modification system and increases transformation efficiency; but crude extract is complete suppressor gene group shift experiment also, and it is included in natural mycoplasma mycoides LC genomic dna and the mycoplasma capri cell (seeing following embodiment 3) separated in the agarose inserted block.
For research inhibition in more detail, do not exist or exist mycoplasma capri or mycoplasma mycoides LC mycoplasma crude extract (as embodiment 2E (ii) in the above (a) in preparation) in situation, the endogenous mycoplasma mycoides LC genomic dna separated in the agarose inserted block (as described preparation in embodiment 2B (ii) in the above), by the fluorescent microscope analysis.As the β-gelase I melting for inserted block of above-mentioned (embodiment 2C (ii)) mycoplasma mycoides LC genomic dna agarose.(1/2000) of 2 μ l dilutions
Figure BDA00002358547600941
Gold nucleic acid gel-colored dose (Invitrogen, Carlsbad, CA) then leniently mixes with the agarose of 5 μ l meltings, and places at room temperature 5 minutes.Mixture is dropped on slide glass and with lid and covers.
Then use and be equipped with Axio
Figure BDA00002358547600942
MRc5 colour camera (Carl Zeiss, Inc, Thorn wood, NY) upright 2 microscopes (Carl Zeiss, Inc) (object lens (100 *); Rhodamine strainer (rhodamine filter)) manifest mycoplasma mycoides LC genomic dna.Obtain fluorescence microscopy images and use the Zeiss from Carl, the AxioVision 4.7.1 version software of Inc. is analyzed.Fig. 7 A illustrates the result of initial treatment agarose inserted block and untreated agarose inserted block.Methylate and the result of Deproteinization step provides respectively in Fig. 7 B-7C, wherein sample and processing are indicated in the above in figure.
As shown in Fig. 7 B, do not exist the natural gene group DNA (demonstration allows to be transferred to recipient cell) processed in the crude extract situation to present dot pattern (right figure), and existing the native gene group DNA (show and suppress to shift) processed in the crude extract situation to form large aggregate (left side two figure).This result shows that the DNA conformation is important for shift experiment.
In order to confirm that conformation is important, as follows with emigrated cells extract after processing by the Proteinase K processing sample.Each yeast inserted block is being supplemented with 1mL Proteinase K reaction buffer [the 100mM EDTA of 40 μ l Proteinase Ks; 0.2% Sodium desoxycholate; 1% sodium lauroyl sareosine; PH8.0] in, 50 ℃ of lower incubations 4 hours.Then, with 1ml 1 * TE damping fluid (20mM Tris-HCl pH 8; 50mM EDTA) rinsing inserted block 4 times, each 45 minutes, and in 0.1 * TE damping fluid, the gentle stirring at room temperature, 2 times, each 30 minutes.After removing final rinsing damping fluid, β for inserted block-gelase I melts and by the fluorescent microscope analysis, as above-mentioned.Result provides in Fig. 7 C, and it shows after incubation together with genomic dna in the agarose inserted block, by Proteinase K, processes and removes crude extract, recovers the initial dot pattern of observing with untreated genomic dna.In addition, as following, described (embodiment 3), Proteinase K is processed the transfer efficiency recovered to a certain extent the natural gene group DNA that processed with crude extract.Therefore, (for example remove deproteinize in cell crude extract, by Proteinase K, process) means like this are provided, the mycoplasma mycoides LC donor gene group DNA separated from yeast host cell by it can be used crude extract to methylate and still successfully be transferred to recipient cell.
Embodiment 2F
Sudden change R-M system (generating mycoplasma capri Δ RE)
Adopt another kind of method as the means that overcome restriction obstacle between host cell and recipient cell.Single restriction enzyme of identifying in mycoplasma capri in the method, (embodiment 2D (i) and table 3 above seeing) is by interrupting the gene coding region inactivation.Restriction enzyme allele enters the genes encoding zone by integration tetracycline resistance marker and is interrupted.
Allos pSD4 spiroplasma citri oriC plasmid (SEQ ID NO:150) is the shown mycoplasma capri (Lartigue, C. etc., Neuclic Acids Res.31:6610-8 (2003)) that transforms.This plasmid is for integrating exogenous array and being effective at mycoplasma capri genome targeted mutagenesis.For inactivation potential restriction enzyme allele (MCAP0050) in mycoplasma capri, use oligonucleotide RE-Mcap-F (the 5 '-gatc that comprises the XbaI site TctagaCtaatgttcaattggatgatata G-3 ' (SEQ ID NO:157)) and RE-Mcap-R (5 '-gatc TctagaCtcaagtcttgtaggagaatc-3 ' (SEQ ID NO:158)), from the interior segments of mycoplasma capri genomic dna amplification gene.The pSD4 plasmid (SEQ ID NO:150) of XbaI cutting is then cut and be cloned into to fragment by XbaI, produce pSD4-Δ Mcap0050.1 (SEQ ID NO:159).Use the scheme of the 5%PEG mediation of describing in embodiment 2C in the above, this plasmid of 10 micrograms is for transforming wild-type mycoplasma capri cell.Select transformant on the solid SP4 flat board that comprises 4 μ g/mL tsiklomitsins.After 37 ℃ of lower incubations 7 days, select several colonies and cell and carried out by propagation go down to posterity for 15 times (15P) in the liquid nutrient medium that comprises 10 μ g/ml tsiklomitsins.Then by the southern blotting technique analysis, clone to verify existing and changing homologous recombination may integrate at the target gene place by single cross of plasmid.
A clone, mycoplasma capri Δ RE clones 1015P, selected, because (as proved by southern blotting technique) its crossing pattern shows to interrupt the MCAP0050 gene by plasmid.Free plasmid do not detected.The cracking extract of whizzer clarification is also by 2D (ii), describing preparation in (b), and is used in during the restriction enzyme of describing in embodiment 2D (ii) tests.Result shows with the wild-type mycoplasma capri and compares, and clones 1015P and does not have restriction enzyme active (data do not show) fully.
Use the middle method of describing of embodiment 2E (ii) in the above, mycoplasma capri Δ RE clone 10 is used as the source that natural mycoplasma capri cell extract is manufactured in the external experiment that methylates.But mycoplasma capri Δ RE clone 10 is not for shifting the good candidate receptor cell of mycoplasma mycoides LC, because it and the mycoplasma mycoides LC donor gene group DNA that exists in yeast comprise identical resistant maker gene (tetM).Therefore, use above-mentioned Strategies For The Cloning, build the another kind of mycoplasma capri Δ RE mutant that carries YCp and puromycin resistance gene rather than tetM gene.From the mycoplasma capri Δ, RE clones 17.5 15P, as 2D (ii) (b) described in the cracking extract of preparative centrifugation machine clarification, and be used in the restriction enzyme of describing test among embodiment 2D (ii).Result shows to be compared with the wild-type mycoplasma capri, in clone 17.515P, does not have the restriction enzyme activity fully.Therefore, select this clone for shifting.
These results show to remove the mycoplasma capri restricted activity from recipient cell and allow the mycoplasma mycoides LC donor gene group DNA separated from yeast, in mycoplasma capri tenuigenin, survive.Alternatively, as above-mentioned, the methylation method provided is used in and shifts the pre-treatment donor dna to protect it.
Embodiment 3
Shift mycoplasma mycoides LC-YCp genomic dna from yeast host cell and enter mycoplasma capri
Top embodiment 1, described and successfully cloned complete bacterial genomes (comprising these mycoplasmas) in yeast host cell.This embodiment has described and successfully shifted the full genome of mycoplasma donor (mycoplasma mycoides LC (the Genbank accession number: NZ_AAZK00000000 of having bred in yeast host cell; GI:149364883)) DNA, enter the mycoplasma recipient cell of different sorts (mycoplasma capri).According to their ramp, select donor and acceptor.But, can also use other mycoplasma donor and acceptors, such as above-described mycoplasma genitalium genome/cell, carry out the method.In addition, the method for describing in this embodiment can for example, be combined with the step (, using all yeast instruments between bacterium donor gene group proliferation period) for when modify in vivo the donor gene group at host cell, then shifts.Therefore, the method can be used for genetic modification donor gene group, such as the genome of the bacterium from having relatively weak exploitation genetic system.
For example, the method can be used for generating the genomic synthetic cell that comprises synthetic transformation.The complete synthetic gene group (Gibson etc., the Science 319:1215-20 (2008) that have synthesized the avirulence bacterial strain of mycoplasma genitalium; Gibson etc., PNAS USA 105:20404-9 (2008)).In that research, in the end, in a step, in yeast, as the kinetochore plasmid, assemble genome.
The method that use provides, such as describe in this embodiment those, in host cell (for example, yeast host cell) this synthetic generation and the natural genome (for example, mycoplasma or other bacterial genomes) of propagation can be transferred to the recipient cell kytoplasm to generate synthetic cell.For example, in order to express the synthetic donor gene group (breeding) of bacterium in yeast in bacterial receptor, can use the method that it is transferred to bacterium (for example, mycoplasma) acceptor from yeast.
Described in embodiment 2 in the above, when from the genomic host cell of propagation donorcells, shifting the donor gene group to different types of recipient cell, the uncompatibility problem appears in different restriction modification systems.Particularly, can determine, when introducing mycoplasma mycoides LC genome and breeding in yeast host cell (above seeing, embodiment 1), by the methyltransgerase of the endogenous expression of these mycoplasmas (seeing top embodiment 2B), unlikely in yeast host cell, expressed.Should determine the fact based on such: methyl transferase gene comprises the UGA tryptophane codon of being processed as terminator codon by the eucaryon yeast host cell.Should determine and show that the mycoplasma mycoides LC donor gene group DNA separated will not methylated from yeast, once and be transferred to the impact that the mycoplasma capri recipient cell can suffer mycoplasma capri recipient cell restriction modification system.Once express after shifting, one or more mycoplasma mycoides LC restriction enzymes are also likely cut the donor gene group.The all respects for overcoming R-M uncompatibility problem of institute's supplying method are described in top embodiment 2.Select to use in these aspects research below, with the donor mycoplasma genome of realizing successfully breeding in yeast, shift and enter different types of mycoplasma recipient cell.
Embodiment 3A
Transfer method
This embodiment described use these technology with successfully from yeast host cell transfer donator mycoplasma genome to the mycoplasma recipient cell.Fig. 8 has shown 3 optional transfer methods, and it can be used for shifting complete genome DNA.The modification of these 3 kinds of methods is used in the embodiment the following describes.The first method (indicating with numeral " 1 " mark arrow in Fig. 8), comprise the agarose inserted block (for example, with β-gelase (melting step)) that digestion comprises genomic dna, then directly is transferred to recipient cell.As shown in FIG. 8, the method typically for example, enters similar cell for transfer donor gene group or nucleic acid from a kind of cell (, donor mycoplasma cell), such as from the mycoplasma donorcells, entering the mycoplasma recipient cell.
The second method (with numeral " 2 " indication) is identical with the first method, except the modified receptor cell with restriction enzyme allele (the Δ RE that suddenlys change; Described in embodiment 2F in the above, generate).Utilize the method, as indicated in Fig. 8, grow in yeast host cell and can successfully be transferred to the mycoplasma recipient cell at the mycoplasma genomic dna separated in the yeast inserted block.Utilize third method (indicating by numeral " 3 " in Fig. 8), sample is methylated and is carried out Deproteinization step (processing with Proteinase K), then melts step (β-gelase digestion) and is transferred to recipient cell.As indication in the drawings, methylate and also help the donor mycoplasma genome that effectively transfer separates after propagation in yeast to enter the mycoplasma recipient cell with the Deproteinization step.As following, described, comparative study comprises and the similar condition of third method, wherein sample under identical condition, do not have a parallel processing in the methylase situation (" simulation methylate (mock methylation) ").These methods and the result obtained by each method are discussed in more detail below.
I. prepare the agarose inserted block
In each method, mycoplasma mycoides LC genomic dna separates (Larionov etc., PNAS USA 94:7384-7 (1997) describes) from yeast strain VL6-48N in the agarose inserted block, as follows.
The yeast cell that comprises mycoplasma mycoides LC genomic dna (as in embodiment 1A, described and generate) is grown under 30 ℃ in selective medium, until OD reaches about 1.5; The compared with control cells that does not comprise the donor gene group is grown under identical condition.Use CHEF mammalian genes group DNA inserted block test kit (Bio-Rad Laboratories, Valencia, CA), the explanation for yeast (Eukaryotic) DNA extraction of recommending according to manufacturers, there is following improvement, prepare each agarose inserted block.Every inserted block is used 6 * l0 9Individual yeast cell (rather than 6 * l0 of test kit recommendation 8Individual cell), to produce every inserted block 6 * l0 8Individual cell, in order to increase the amount of the available mycoplasma mycoides LC of each inserted block genomic dna.After cell embedding is in the agarose inserted block, for the peptic cell wall, use Zymolyase TM100T enzyme (USB Corporation, Cleveland, OH) replaces lyticase (Bio-Rad Laboratories, Valencia, CA).Zymolase TMEnzyme adds the inside or the outside of inserted block to the concentration of 5mg/mL, and places 2 hours under 37 ℃.
1 * TE damping fluid (20mM Tris-HCl, pH 8; 50mM EDTA) after middle rinsing, by 5ml Proteinase K reaction buffer [the 100mM EDTA with being supplemented with every ml inserted block 200 μ l Proteinase Ks; 0.2% Sodium desoxycholate; 1% sodium lauroyl sareosine; PH 8.0] at 50 ℃ of lower incubations twice 24 hours, the yeast cell of cracking embedding digesting protein.The agarose inserted block then at room temperature uses 40ml 1 * TE damping fluid to stir rinsing 4 times, and each 1 hour, and be kept in identical damping fluid under 4 ℃.For the yeast inserted block (below seeing) digesting with restriction enzyme subsequently, add 1mM phenylmethylsulfonyl fluoride (PMSF) between the rinsing clear-up period for the second time.
Cleaning
Before these researchs, do not know whether the pastoris genomic dna separated together with the mycoplasma mycoides LC donor gene group DNA extracted can affect or abolish shift reaction from yeast.Therefore, utilize the method, prepared 2 groups of donor gene group DNA, one of them experiences optional " cleaning " step after peptic cell wall and Proteinase K processing.Design " cleaning " step to remove pastoris genomic dna from sample.
" cleaning " sample is also then cleared up little cerevisiae dna fragment by electrophoresis by the restriction enzyme mixture process of specificity digesting yeast genomic dna, or directly application of sample to the pulsed field sepharose separately to be retained in ring-type genome in well and the linear yeast chromosomal (Lartigue etc. to the well outside by electrophoresis, Science317,632 (2007)).
In order to clear up with enzyme mixture, the yeast inserted block is at 1ml 0.1 * TE damping fluid (2mM Tris-HCl pH 8.0,5mM EDTA) in, rinsing is 2 times, each 1 hour, be supplemented with 1 * NEB damping fluid 2 (New England Biolabs of BSA at 1ml, Ipswich, MA) in balance 1 hour, and for the genomic dna existed in inserted block, 50 unit limit enzyme mixtures (AsiSI, RsrII and FseI) digest and spend the night in 100 μ l reaction volumes.The yeast inserted block is at room temperature used (20mM Tris-HCl pH 8.0, the 50mM EDTA) rinsing 1 hour of 1mL 1 * TE damping fluid and is loaded onto (120 minutes, 120 volts) in 1% TAE sepharose.The agarose inserted block shifts out and preserves under 4 ℃ from well.
In order only to clear up by electrophoresis, other yeast inserted blocks are in TAE 1 * middle 1%LMP gel, with clamper uniform electric field (6) (CHEF DR III; Bio-Rad) carry out electrophoresis.Burst length slow is raised to 120 seconds from 60, under 3.5V/cm 24 hours.After migration, inserted block shifts out from well, under 4 ℃, is kept in 1 * TE damping fluid.
Ii. general transfer method
For every kind of method, shift and to carry out as follows, except for third method, before melting with β-gelase, sample is methylated and the Proteinase K processing, described in embodiment 3A (iii) below.
A. recipient cell
Ten two (12) mL mycoplasma capri recipient cells (wild-type or Δ RE (as generated in embodiment 2F)) are being supplemented with foetal calf serum (17%), glucose (10g/L), 2ml phenolsulfonphthalein (1%) and 100 μ l penicillin Gs (5mg/ml)) SOB (+) substratum (Bacto SOB substratum (Becton Dickinson; Franklin Lakes, NJ) middle growth, until the pH of culture reaches pH 5.7 to 5.85 (about 5 * 10 7Individual cell/ml).Recipient cell under 10 ℃, at 4575 * g centrifugal 15 minutes, (10mM Tris-HCl, pH 6.5 in 6mL S/T damping fluid; With 250mM NaCl) in rinsing once, be resuspended in 400 μ l CaCl 2(0.1M) in and incubation on ice 30 minutes.
B. the donor gene group DNA that preparation separates in the agarose inserted block
Before transfer, as embodiment 3A (i) in the above (a) described in, the agarose inserted block that comprises mycoplasma mycoides LC genomic dna from the yeast preparation is gentle the stirring at room temperature, at 0.1 * TE damping fluid [2mM Tris-HCl, pH 8.0,5mM EDTA] in rinsing 2 times, each 30 minutes.Remove damping fluid fully and under 65 ℃, with 10 * β of 1/10 volume-gelase damping fluid [10mM Bis Tris-HCl pH 6.5; 1mM Na 2EDTA] melt agarose inserted block 10 minutes.The agarose melted is cooled to 42 ℃, continue 10 minutes, and, at this temperature, the β of every 100 μ l inserted blocks and 3 units-gelase I (New England Biolabs, Ipswich, MA) is incubated overnight together.As embodiment 3A (i) below (c) described in, β in third method-gelase is methylated before processing and Proteinase K is processed (Fig. 8 (" 3 ")).
C. shift existing in the 5%PEG situation
On ice after 30 minutes, 400 μ l recipient cells comprise with 800 μ l the donor gene group DNA agarose inserted block that 100 μ l dissolve---as generated in embodiment 2B (ii) in the above---SP4 (-) substratum leniently mix.As following, just, before carrying out next step, add inserted block.
1300 (1300) μ l 2 * fusion damping fluid (20mM Tris-HCl pH6.5,250mM NaCl, 20mMMgCl 2, 10% PEG-6000 from Fluka] add to immediately in the mixture of SP4 (-), genomic dna and cell and by leniently shaking pipe and make mixture even in 30 seconds.Under 37 ℃, after 50 minutes, add the SP4 of 10ml pre-warm cell mixing leniently.Under 37 ℃, after another 3 hours, under 10 ℃, with 4,575 * g eccentric cell 15 minutes, be resuspended in 1.2ml SP4 and bed board on the SP4 flat board that comprises 4 μ g/ml tsiklomitsins and 150 μ g/ml X-gal (2 flat boards of each sample).After 3-4 days, select each colony and grow in the broth culture that comprises 10 μ g/ml tsiklomitsins.
Iii. methylate and protease K digesting (details of third method)
For third method (Fig. 8, " 3 "), before melting with β-gelase, methylate from the mycoplasma mycoides LC genomic dna of yeast agarose inserted block, then the Deproteinization step.For this process, inserted block is at 200mM Tris-HCl pH 7.5; In 50mM EDTA, rinsing is 2 times, and 30 minutes, and at the damping fluid that methylates (100mM Tris-HCl pH 7.5; 10mM EDTA, 3 μ M DTT, 200 μ M S-adenosylmethionines) in gentlely stir balance 2 times 30 minutes.After balance, the yeast inserted block is divided into 4, and adds 100 μ l methylation reactions (1 * damping fluid methylate transferring enzyme methylates) to and 37 ℃ of lower incubations 16 hours.For 100 μ l reactions, 5 μ l wild-type mycoplasma mycoides LC cell extracts, or the mycoplasma mycoides LC specificity methyltransgerase (M.GANTC, M.CCATC, M.GCATC, M.CCTTC, M.III type) of 7.5 μ l mycoplasma capri Δ RE (clone 1015P) cell extract (about 125 μ g albumen) or 2.5 each purifying of μ l.Each cell extract is as described the ground preparation in embodiment 2E (ii) in the above, and the methyltransgerase of purifying is as described the ground preparation in embodiment 2E (i) in the above.The methylation reaction of the methyltransgerase that comprises mycoplasma mycoides LC cell extract or purifying also is supplemented with 5 μ l dam methyltransgerases (New England Biolabs, Ipswich, MA).
After methylating, each yeast inserted block is being supplemented with 1mL Proteinase K reaction buffer [the 100niM EDTA of 40 μ l Proteinase Ks; 0.2% Sodium desoxycholate; 1% sodium lauroyl sareosine; PH8.0] in 50 ℃ of lower incubations 4 hours.Inserted block is then with 1ml 1 * TE damping fluid (20mM Tris-HCl pH 8; 50mM EDTA) rinsing 4 times 45 minutes at room temperature gentlely in 0.1 * TE damping fluid stir rinsing 2 times 30 minutes.After removing final rinsing damping fluid, β for inserted block-gelase I melts, and as in the above, (b) part is described, and then shifts.
Embodiment 3B
The research that the method that showing to use provides successfully shifts
Use the general method of describing in embodiment 3A in the above, under the condition of listing in the following Table 11, carry out shifting mycoplasma mycoides LC Genome donor DNA and enter wild-type and restriction enzyme-defect mycoplasma capri recipient cell from yeast host cell." untreated " sample, before melting, was not both methylated, and with Proteinase K, did not process yet." simulate-methylate " sample incubation under the same terms with the sample that methylates, there is no enzyme or cell extract.Methylate to process and comprise with donor or recipient cell extract and methylate and use the methylase of purifying to methylate.
Table 11: jump condition
Figure BDA00002358547601001
By under 37 ℃, be chosen in the blue colony of growth on the SP4 substratum that comprises tsiklomitsin, score to result, and show in 12A below being provided at.As pointed out in this table, in order to methylate with the mycoplasma mycoides extract and to be transferred to wild-type cell (sample 2), by digesting with yeast specific enzymes (b) or passing through electrophoresis (c), some samples are cleared up to remove cerevisiae dna, as above-mentioned.
Table 12A: by the donor phenotype, quantize to shift colony
Figure BDA00002358547601011
aAt least 3 experiments average.The error of report is average deviation.
bUse AsiSI, RsrII, FseI mixture restriction enzyme scheme, the yeast inserted block has been eliminated pastoris genomic dna.
cUse the pulsed field gel electrophoresis scheme, the yeast inserted block has been eliminated pastoris genomic dna.
As shown in showing in 12A in the above, in sample, obtain to the colony of mycoplasma mycoides LC phenotype similar (according to similar outward appearance and growth velocity) (as Lartigue etc., (2007) Science 317 632-8 (6) describe), described sample is used the methylase of mycoplasma mycoides LC extract or purifying methylate and be transferred to ( sample 2,3,7 and 8) in wild-type or Δ RE recipient cell.The anti-restriction enzyme produced by mycoplasma mycoides LC of the mycoplasma mycoides LC donor gene group that this results verification has been bred in yeast and the enzyme that produced by the recipient cell system the two.
On the contrary, simulate-methylate and untreated mycoplasma mycoides LC genome only shifts in the situation that mycoplasma capri Δ RE recipient cell (sample 6 and 10) produces donor-phenotype colony, and in the situation that wild-type receptor cell (sample 1 and 5) do not produce.For example, when simulation process be transferred to mycoplasma capri Δ RE recipient cell (sample 6 and 10) with untreated donor gene group DNA the time, obtain respectively 34 and 37 colonies.But, when mycoplasma mycoides LC genomic dna simulation process or untreated is transferred to wild-type mycoplasma capri recipient cell (table 12A), do not obtain colony.
Similarly, obtain colony (32 and 9 colonies, table 12A) in the two kinds of recipient cell types (sample 4 and 9) that have by the donor gene group of mycoplasma capri extract-treated.Because the mycoplasma capri extract provides the protection of anti-mycoplasma capri restriction enzyme, it is important for successfully transform the mycoplasma mycoides LC donor gene group of breeding in yeast that this result shows to avoid mycoplasma capri acceptor restriction system.Following truth: inactivation mycoplasma capri restriction system (mycoplasma capri Δ RE recipient cell) enough allows successfully to shift and activates mycoplasma mycoides donor gene group, shows that inactivation this receptor cell restriction system is also enough for these events.Therefore, donor mycoplasma mycoides LC restriction system (when being transferred to recipient cell, it is activated) does not show and forms the obstacle from yeast transfer donator genome to the mycoplasma capri recipient cell.
Reclaim the existence that colony depends on the mycoplasma capri recipient cell in shift experiment, as when recipient cell is omitted from reaction, do not observe colony.And shift experiment also depends on the mycoplasma mycoides LC genome from yeast, as when only from the pastoris genomic dna of yeast or YCp plasmid, being used as donor dna, do not observe colony.The recipient cell colony that the southern blotting technique confirmation has received the donor gene group from yeast is mycoplasma capri and mycoplasma mycoides LC genotype.
There is no " cleaning ", cerevisiae dna exists in comprising the sample of genomic dna.Purifying donor gene group DNA from pastoris genomic dna (show in the above in 12A by " b " and " c " indication) is not enough to change and shifts result, shows that acceptor mycoplasma capri cell can tolerate exist (table 12) of non-specific or carrier DNA.In addition, use the donor gene group DNA separated from 2 different yeast strains (VL6-48N and W303a) to obtain positive transfer result, show that the genotype of host yeast strain and/or phenotype may be inessential for shift experiment.
These results shown together the mycoplasma capri cell activation from the mycoplasma mycoides LC genome of dormancy on the phenotype of yeast, cause generating the ability of the mycoplasma mycoides LC cell of living, and the method therefore provided can be used for shifting the full genome of protokaryon of growing in eukaryotic host cell and enters and donor or the different types of acceptor prokaryotic cell prokaryocyte of host.
Shifting non-methylated mycoplasma mycoides LC genome from yeast enters mycoplasma capri Δ RE recipient cell and confirms that the recipient cell that transforms, selects comprises the donor gene group and not have to be selected owing to lacking some mycoplasma mycoides LC components rather than genomic dna.This is confirmed to be due to following truth: between transition phase, except genomic dna, do not have mycoplasma mycoides LC cell or component.
In another kind of embodiment, YCpMmyc1.1, and the YCp genome (YCpMmyc1.1-Δ typeIIIres::URA3 and YCpMmyc1.1-Δ typeIIIres) of transformation also separate from yeast strain W303a.Shift all 3 YCp genomes and enter the blue colony of tetracyclin resistance (table 12B) that the mycoplasma capri recipient cell produces similar amt.Large deletion clone above-mentioned (YCpMmyc1.1-Δ 500kb) is as suitable contrast, because but it lacks the indispensable gene of many suppositions still retains YCp element and tetM.As expected, when this genome is transferred to the mycoplasma capri recipient cell, do not reclaim colony.
Reclaim colony and depend on the two the existence of mycoplasma capri recipient cell and mycoplasma mycoides genome in all these shift experiments.The donor YCp genome that comprises pastoris genomic dna is used in experiment described herein.But purifying donor YCp genomic dna is not enough to change to shift result from pastoris genomic dna, it shows that acceptor mycoplasma capri cell can tolerate exist (the table 12B) of non-specific or carrier DNA.Use, from 4 of the bacterial strain V16-48N donor YCp genomic dnas of transformant culture and 4 W303a strains separation independently, obtains positive transfer result.Therefore, bacterial genomes can stably be cloned in two yeast strains.By using mycoplasma mycoides specificity IS 1296 elements to carry out DNA marking analysis (data do not show) as probe, confirm that the colony reclaimed is mycoplasma mycoides.Show, by using III type restriction gene sequence as the southern blotting technique analysis (data do not show) of probe with by sequenced genes seat (Figure 17), III type restriction gene is lacked in the engineering bacterium by PCR.
Table 12B
Figure BDA00002358547601031
* by the digestion of the mixture with Asi SI, RsrII and FseI, subsequently by pulsed-field gel electrophoresis, the yeast inserted block is removed pastoris genomic dna.
Figure BDA00002358547601032
Remove pastoris genomic dna by using pulsed-field gel electrophoresis yeast inserted block.
Table 12B provides from yeast and has shifted the result that mycoplasma mycoides YCp genome enters wild-type and RE (-) mycoplasma capri recipient cell.Counting shifts mycoplasma mycoides YCp genome from yeast and enters the blue colony of the tetracycline resistance obtained after the mycoplasma capri acceptor.The method that use is described in Fig. 8 carries out the wild-type mycoplasma capri and mycoplasma capri RE (-) shifts.For untreated sample, with β-gelase digesting yeast inserted block (melting step) and be transferred to two kinds of recipient cells.The sample of processing was methylated and was processed with Proteinase K before melting step.Simulate-methylate sample and methylate the sample same treatment, except not adding the methyltransgerase of extract or purifying.The VL6-48N yeast agarose inserted block used in this experiment carries YCpMmycl.l.W303 yeast agarose inserted block carries YCpMmycl.l or YCpMmycl.l-Δ 500kb, and YCpMmycl.l transforms in yeast (YCpMmycl.l-Δ typeIIIres::URA3 or YCpMmycl.l-Δ typeIIIres).The quantity that shifts body is at least the average of 3 experiments.The error of report is absolute mean deviation.
Embodiment 4
Modify the donor gene group in yeast host cell
This embodiment has described for seamless modification being introduced to the use of the method for the mycoplasma donor gene group that has been cloned into yeast host cell.
Yeast---yeast saccharomyces cerevisiae---has been developed to can clone large DNA fragmentation as the two host of linear and saccharomyces annulatus artificial chromosome (YAC).Once be cloned into yeast, but the genetic tool of these YAC Application standards operation.Shift the DNA of these modifications to being applicable to the host cell of expressing, allow the functional study of gene and their regulation and control.The full genome of cloning bacteria and shift subsequently such genome and get back to their initial cell environment in yeast, made this application expand to genomic level from gene level.
Utilize two step reorganization schemes of anti-selective marker to be used in yeast to modify YAC (Tucker and Burke (1996) Nucleic Acids Res, 24,3467-3468).In these methods, at first anti-selective marker is recombined into YAC and selects.Next, the new reorganized replacement mark of DNA fragmentation that comprises the expectation change, it is again selected.The mark the most often used in these processes is the URA3 gene, and it recovers the uridylic autotrophy in the defect bacterial strain.By with 5-fluororotic acid (5-FOA), process the anti-selection carry out URA3 gene substitution (Boeke etc., (1984) Mol Gen Genet, 197,345-346).The first, the method is recovered uracil auxotrophy, and it can be then by the modification for advancing to take turns again.The second, the method has produced seamless modification.This basic skills for seamless modification is enhanced in many aspects.Delitto perfetto method by utilize endonuclease I-SceI near the target gene seat, introduce double-strand break (DSB) (Storici etc. (2001) Nat Biotechnol, 19,773-776).The formation raising homologous recombination repair efficiency numbers order of magnitude of DSB (Storici etc. (2003) PNAS USA, 100,14994-14999).Another kind method, being called series connection and repeating to give prominence to, target site flank manufacture series connection repeat order (Akada etc. (2006) yeast, 23,399-405).The method only needs once to transform, and then 5-FOA is counter selects, and additive method needs to transform for 2 times.These methods can be applicable to disappearance, point mutation or gene substitution.
In yeast assembling and clone as the synthetic mycoplasma genitalium genome of ring-type YAC by (Science, 319,1215-1220 such as Gibson; With Gibson etc. (PNAS USA, 105,20404-20409 (2008)), describe.Yeast can be used as the platform of synthetic bacterial genomes in directly transformation or redesign body.
As each chapters and sections are described below, carry out 5 different locus specificity modifying method, comprise single base cytidine disappearance (309 to be modified in the genomic CDS139 locus of synthetic sMgTARBAC37 mycoplasma genitalium that is introduced into and is retained in yeast host cell, 388) target region (target gene seat), as Gibson etc., Science 319,1215 (2008) and the US publication 20090275086 of Gibson etc. describe, and as in the above described in embodiment 1C.
Wine brewing yeast strain VL6-48N (MAT α his3-Δ 200 trp1-Δ l ura3-52 lys2 ade2-101 metl4) and W303a (MATa ade2-l ura3-l his3-ll, 15 trpl-1 leu2-3,112 canl-100RAD5) carry as above-mentioned Gibson etc., Science describes and the synthetic gene group of embodiment 1 in providing in the above.Yeast cell (the Amberg etc. that grow in standard rich medium (YEPD) and synthesis of glucose (SD) or synthetic semi-lactosi (SG) minimal medium, (2005), " method in yeast genetics: cold spring harbor laboratory's procedures manual (Methods in yeast genetics:A cold Spring Harbor Laboratory Course Manual) ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.230).
Run through the primer nucleic acid sequence of using in the various researchs of this embodiment (embodiment 4) description lists in table 13.Dash in primer sequence means that the structure of primer is chimeric.With the homeologous primer sequence part of mycoplasma genitalium, with the lowercase type, list.With the primer sequence part of mycoplasma genitalium homology, with the capitalization type, do not list.I-SceI cleavage site (below seeing) goes out with the underscore text column.In the method for describing at this embodiment, all primers are customization synthetic (Integrated DNA Technologies (IDT)).Length is greater than the primer of 60bp by the polyacrylamide gel electrophoresis purifying.
According to disclosed method (Gietz etc., Nucleic Acids Res, 20,1425 (1992)) use the lithium acetate integration transformation, use 2-3 μ g PCR product and 25 μ g carrier DNA (Salmon testis DNA, Sigma), the PCR construct is introduced and comprised in the genomic yeast strain of mycoplasma genitalium.
Table 13: the primer used in mutagenesis research
Figure BDA00002358547601051
Figure BDA00002358547601061
Figure BDA00002358547601071
Embodiment 4A
Use the modification of conventional two step homologous recombination methods
Use conventional two step homologous recombination method (Rothstein, R.Methods Enzymol, 194,281-301 (1991)) introduce mutation site-specific, to proofread and correct the single base cytidine disappearance in the synthetic mycoplasma genitalium genome retained in yeast.The method schematic illustrations is in Figure 12 A.
I. passing through homologous recombination---the URA3 gene is introduced in the conventional sequence displacement
The primer that use is listed in table 13, carry out ordinary method (conventional sequence displacement) as follows.In comprising two first steps that transform continuously,---its sequence on table 13 in list---(is described in R.S.Sikorski and P.Hieter from plasmid pRS306 to use primer URA-F and URA-R, Genetics 122, in 19 (1989)) and pcr amplification URA3 gene (1,066bp).The URA3 gene of this amplification comprises two 50-bp end sequences identical with the part of part mycoplasma genitalium genome area for the target that suddenlys change.Use archaeal dna polymerase (Takara, Madison, WI), under the condition of recommending in manufacturers, carry out the PCR reaction.Use the lithium acetate integration transformation, the PCR product is introduced and comprised in the genomic yeast strain of mycoplasma genitalium.
Select independent Ura+ transformant, and by using the diagnostic primers Seq-F and the Seq-R that list in table 13 in the above to carry out pcr analysis, with the URA3 gene of confirming amplification in the donor gene group correct position insert.Seq-F and Seq-R primer are at target gene seat (adorned target gene group zone) flank, and separate 0.4kb along genome, therefore from the genome amplification that comprises the URA3 token-replacement, will produce 1.35kb PCR product (being illustrated schematically in Figure 12 A).On sepharose separately, it is visualized to verify correct insertion URA3 gene to the product of PCR reaction.
Ii. second take turns conversion: introduce wild-type fragment and select
Take turns conversion for second, by using the primer Amp-F that lists in table 13 in the above and the pcr amplification of Seq-R, produce 328bp wild-type DNA fragmentation (with the portion homologous of target region but do not comprise CDS 139 locus list base deletions).This fragment is introduced into the URA3 displacement bacterial strain that uses lithium acetate integration transformation method to obtain from the first round.
Second take turns conversion after, cell under 30 ℃ in the dull and stereotyped grow overnight of SD-HIS, to exhaust residual Orotidine-5 '-in losing any yeast cell of URA3 gene '-phosphate decarboxylase (by the URA3 genes encoding).Use is supplemented with the SD substratum (the SD-HIS flat board that comprises FOA) of 5-fluororotic acid (FOA) the URA3 gene is lost and selected (Boeke etc., Mol Gen Genet, 197,345-346 (1984)).
Use diagnostic primers Seq-F and Seq-R (top description is also listed in table 13), the PCR of the genomic dna by the clone from selecting, estimate the correction (exactness) of sudden change.Use Takara archaeal dna polymerase (Takara, Madison, WI), under the condition of recommending in manufacturers, carry out the PCR reaction.The PCR product separates on sepharose.Use these primers, the genomic dna that amplification comprises CDS 139 locus (or have the initial gene seat of single base deletion or have the wild-type sequence of displacement) will produce the PCR product (data do not show) of 0.4kb DNA fragmentation.
Test 97 FOA resistance colonies by the method.All result is summarised in the first row of table 14; As shown in table 14, by the pcr amplification genomic dna, do not have FOA resistance colony to produce correct 0.4kb fragment.This result shows not occur accurate homologous recombination (HR) between the wild-type DNA fragmentation entered and target site.But the disappearance of not expecting has caused the loss of URA3 mark in these FOA resistance colonies.Consider in yeast the mycoplasma genitalium genome as ring-type YAC propagation, except Histidine prototroph, its host is not had to supplementing of function, this possibility is possible.Therefore, the disappearance in the donor bacterium genome and rearrangement may be neutral to the existence of host cell.
Iii. multiplex PCR
In order by being evaluated at, to select this possibility of the genomic integrity detection of mycoplasma genitalium in yeast, according to Gibson etc., PNAS USA, 105 (51): 20404-9 (2008) and Gibson etc., Science 319,1215 (2008) descriptions, carry out multiplex PCR (MPCR).According to disclosed scheme (Kouprina and Larionov, Nat.Protoc.3,371 (2008)), as described in embodiment 3, carry out total DNA separation for pcr analysis from yeast in the above.(scope is from 125bp to 1025bp to produce 10 amplicons to be designed for the primer sets (organizing 3) of MPCR, with 0.1kb, increase progressively), it is around the about every 60kb DNA distribution (Gibson etc. of mycoplasma genitalium genome, PNAS USA, 105 (51): 20404-9 (2008)).Use the multiple PCR reagent kit of Qiagen (Valencia, CA) to carry out multiplex PCR.10 * primer the raw material that comprises 20 kinds of oligomers that 1/50 volume (2 μ l) DNA extraction thing and 1 μ l comprise every kind of 5 μ M in each 10-μ l reaction.Loop parameter is 94 ℃, 15min, and then 35 circulate 94 ℃, 30 seconds, 52 ℃, 90 seconds and 72 ℃, 90 seconds, then under 72 ℃, carry out 3 minutes incubations of single.Follow each reaction application of sample to 2% E-gel (Invitrogen) of 2 μ l and apply 72V 30 minutes.Use Amersham Typhoon 9410 fluoroscopic image instrument video picture bands.
Result shows each for 22 FOA-resistance colonies, and the amplification of total DNA does not produce all 10 amplicons.The MPCR of any FOA-resistance clone does not produce 2 amplicons (together with in the mycoplasma genitalium genome, they cluster) of length 0.55 and 0.65kb.CDS 139 target gene seats are positioned at 3kb place, 0.65kb amplicon upstream (the gel result does not show).
In the mycoplasma genitalium genome that this result shows to breed in yeast, non-specific disappearance or rearrangement have occurred.In these clones the loss (proof by selection by them with FOA) of URA3 mark may come from the mycoplasma genitalium genome in homologous recombination between tumor-necrosis factor glycoproteins.Due to this restructuring, wherein the cell of URA 3 mark disappearances can be survived on the FOA substratum.Therefore, utilize this nconventional method, non-specific URA3 gene is lost, and the recombination event displacement URA3 than the wild-type DNA fragmentation intention of introducing by utilization, have higher probability.
This problem schematic illustrations in Fig. 9 of conventional modifying method, wherein wild-type fragment is introduced (Fig. 9 A) (as generated in embodiment 4A in the above) in the yeast that carries the mycoplasma genitalium with URA3 insertion, then on the SD-HIS flat board that comprises FOA, select, produce and select 2 dissimilar recombination event (Pl (restructuring between wild-type fragment and genome) and P2 (restructuring between repeating in genome)) (Fig. 9 B).These events are carried generation in the cell that is illustrated in the optional product in Fig. 9 C.Because the mycoplasma genitalium genome comprises a plurality of repetitions, so the probability that the likelihood ratio of the non-specific loss of URA3 gene (P2) is lost due to the restructuring (P1) of intention is larger.The disappearance that general non-specific loss may observe due to any clone who comprising of using this ordinary method to cause is intended to sequence.These results show for modify the improved method of donor gene group needs in yeast host cell.
Table 14:FoA +The quantity of correct sequence displacement and complete mycoplasma genitalium amplicon in the clone
Figure BDA00002358547601091
Embodiment 4B
Optional seamless modifying method
Two kinds it is reported that more effectively other seamless modifying method are used, to attempt to be modified at the identical target region of synthetic mycoplasma genitalium donor gene group in yeast, as above-mentioned.
i.Delitto?perfetto
Two kinds it is reported the first of effective means, and delitto perfetto (Storici, F. etc., Nat Biotechnol, 19,773-776 (2001)) schematic illustrations is in Figure 10 A.In the method, introduce double-strand break in the target DNA site and improve the several orders of magnitude of restructuring.
Delitto perfetto method used herein is used construct like this: its CORE box flank have with the 50bp sequence of target gene seat upstream region homology and with the 50bp sequence of target gene seat downstream area homology, described CORE box comprises: the nucleotide sequence of being identified by the specific nucleic acid restriction endonuclease, inducible promoter, inducible promoter are controlled gene and the selection/anti-selective marker (Figure 10 A) of lower coding specific nucleic acid restriction endonuclease.Therefore, design this box, once in order to induce the endonuclease expression of enzymes, endonuclease cuts at its recognition site place in box, and generating double-strand break also increases recombination efficiency in the site of expectation.
A. generate dellitto perfetto box
By merging PCR so that comprise GAL1 promotor (evoked promoter) and the first fragment of I-SceI gene (endonuclease-encoding gene), with URA3 (selection/anti-selective marker) gene fragment, merge and generate delitto perfetto mutagenesis-cartridge.Use the primer URA-F and the URA-R that list in table 13 in the above, as in embodiment 4A in the above, describe amplification URA3 gene fragment from plasmid pRS306 (1,066bp).Primer GaI-F and GaI-R that use is listed in table 13, (be described in Storici etc., PNAS USA, 100 from plasmid pGSKU, in 14994-14999 (2003)) increasing comprises 1 of GAL1 promotor and I-SceI gene (GAL1 II-SceI gene fragment), 184bp fragment.Use basically as at Shevchuk etc., Nucleic Acids Res, the recombinant PCR technology of describing in 32, el9 (2004) is merged PCR.
B. the first round transforms
Use the lithium acetate integration transformation, box is introduced and comprised in the genomic yeast strain of mycoplasma genitalium.Select independent Ura +The diagnostic primers Seq-F that transformant using is listed in table 13 in the above and Seq-R (in Figure 10 A shown in little single arrow of insertion point flank), by pcr analysis, with confirm gene in the donor gene group correct position insert.The genome that uses these primer PCRs to comprise to insert box will produce the product of 2.5kb.Product from the PCR reaction separates on sepharose, and it is manifested to verify correct insertion URA3 gene.
C. induce endonuclease expression of enzymes and second to take turns conversion
In the PCR with diagnostic primers reaction, test is positive be cloned in the SD/ semi-lactosi/-the HIS substratum in growth 4 hours, with abduction delivering I-SecI endonuclease, it is controlled by the GAL1 promotor and therefore expresses when comprising when semi-lactosi is grown in as the substratum of sole carbon source.The endonuclease expression of enzymes of inducing is intended, by cutting just at the 18bp in the regional downstream with genome homology recognition sequence, producing double-strand break in box.After inducing, the wild-type DNA fragmentation is transformed into cell, described in embodiment 4A in the above.
Cell is grown on the SD-HIS flat board that comprises FOA, to select to have lost the cell of URA3 mark, described at embodiment 4A.Used the diagnosis PCR of Seq-F and Seq-R primer, with the FOA resistant cell of determining selection, whether comprised the genome that wherein box has been replaced by wild-type fragment (it will produce the PCR product of 400 base pairs).As indicated in table 14 in the above, in the FOA resistance isolate of 60 tests, neither one produces the amplicon of correct size.Result shows that the mycoplasma genitalium genome from 60 FOA resistance isolates comprises coarse CDS 139 locus disappearances.
Ii. series connection repeats to give prominence to
The seamless deletion method of the second report, series connection repeats to give prominence to, the accurate excision based on by the nucleic acid fragment of homologous recombination (HR) between two tandem repetitive sequences, and at Akada etc., Yeast, describe in 23,399-405 (2006).This technology can be suitable for gene substitution.Utilize the method, carry out the seamless disappearance of CDS 139 locus in comprising the genomic identical yeast strain of mycoplasma genitalium, rather than proofread and correct single base deletion.
A. generate series connection repetition box by merging PCR
Generate fusion product, its comprise the URA3 mark and with just in the homeologous 358bp fragment (" repetition " fragment) (being labeled as the large arrow of " repetition " in Figure 10 B) of the upstream of target gene seat.The same procedure that use is described in embodiment 4A, used Ura-F and Ura-R primer (table 13), by PCR, produces 1,066bp URA3 labeled fragment.Be used in Amp-F and the Seq-R primer listed in table 13, by pcr amplification, produce the 358bp repeated fragment.
The recombinant PCR technology of using as describing in above-described embodiment 4B (i), by merging PCR, connect two portions as follows.At first, each that list in table 13 in the above comprises chimeric fusion primers F usl and the Fus2 with the part of URA3 gene and " repetitions " fragment homology, be used in PCR with amplification URA3 gene and in another PCR with the repeated fragment that increases.Comprise the zone with other reaction product homologies from the product of each reaction, because share the overlapping homologous sequence of 40 base pairs altogether between two amplified productions.Product then carries out the PCR circulation, there is no primer, utilizes low annealing temperature to connect product, produces the fusion product that comprises junction fragment.
In order to generate final mutagenesis-cartridge (seeing Figure 10 B), fusion product is used the chimeric primers UM2-70 that lists in table 13 in the above and MUT-70 to carry out PCR and increases.As shown in that table, 50 base pairs (bp) homologue (5 ' end of the homologue that each of these primers comprises fusion product and target region; Lowercase).Gained box (being illustrated in Figure 10 B) comprises the 50bp homologue of 50bp homologue (upstream of single base deletion), URA3 mark, repetition box and the target region 3 ' part of 5 ' part of target region in the following order.With this direction design box, once, in order to be transformed into yeast host cell, with 450 base pair target regions in the genomic CDS139 locus of this box displacement (passing through HR) mycoplasma genitalium, can in genome, produce the zone (being labeled as the large arrow of " repetition " in Figure 14 B) that comprises two tandem repetitive sequences by the flank in the URA3 selective marker.Comprise that tandem repetitive sequence helps disappearance (giving prominence to) box with the homologous recombination by between two tumor-necrosis factor glycoproteinss.This event can be removed the URA3 mark also can be by growing and selected in comprising the substratum of FOA.
B. transform and analyze
Use the lithium acetate integration transformation, box is introduced and comprised in the genomic yeast strain of mycoplasma genitalium.Select independent Ura +The diagnostic primers Seq-F that transformant using is listed in table 13 in the above and M2-detl-R (in Figure 10 B shown in little single arrow of insertion point flank), by pcr analysis, with confirm gene in the donor gene group correct position insert.Utilize the PCR of the wild type gene group of these primers will produce the 1kb product.On the other hand, comprise the Genomic PCR that inserts box and produce the 1.973kb product.Product from the PCR reaction separates on sepharose, and it is manifested to verify correct insertion URA3 gene.
Detect positive cell by the PCR to correct insertion and grow on the SD-HIS flat board that comprises FOA, to select to lose the cell of URA3 mark, described at embodiment 4A.Used the diagnosis PCR of primer Seq-F and M2-detl, whether comprise wherein box with the FOA resistant cell of determining selection and lacked the 450 base pairs seamless disappearance partly that stays target region.Utilize these primers, to the accurately Genomic PCR of disappearance has wherein occurred, will produce the 0.55kb product.As indicated in table 14 in the above, in this pcr amplification, in 38 FOA resistance isolates, neither one produces product.
As described, the DNA from 9 FOA resistance isolates is carried out to MPCR in embodiment 4A.As indicated in table 14, in these 9 separation, only have 1 to produce complete copy (all 10 products) in the above.Do not exist the complete copy sublist bright, between the tumor-necrosis factor glycoproteins in donor gene group self, restructuring has occurred.This result shows that the recombination frequency between the tandem repetitive sequence of URA3 mark flank is more much lower than the restructuring between tumor-necrosis factor glycoproteins in the mycoplasma genitalium genome.
Generally speaking, come the result of comfortable embodiment 4A and this embodiment (4B) institute descriptive study to show, currently known methods based on the URA3/FOA system is not enough to operate and transform in these yeast host cells mycoplasma genitalium donor gene group in this particular system, and the most of FOA resistance colony reclaimed in these researchs is by the non-specific loss of undesirable recombination event URA3 mark during operating process, and showing need to be for modifying improving one's methods of donor gene group at yeast host cell.
Embodiment 4C
Use the modifying method of series connection repetition and endonuclease cutting (TREC)
Result based on studying described in embodiment 4A and 4B, the recombination frequency between the deducibility series connection that (embodiment 4B (ii)) wishes in method for protrusion repeats can improve by introduce double-strand break near the target gene seat.Provide such method (TREC), it uses series connection to repeat and endonuclease cuts (TREC) and is used in modification donor nucleic acid in yeast host cell.This embodiment the method for using this to provide has been provided has been modified at identical target gene seat in mycoplasma genitalium donor gene group in yeast.Result of study confirms that repeating to introduce the ds fracture by the series connection in this system near target site has increased recombination efficiency.
Method of design is lost to reduce non-specific background when selecting the URA3 mark is counter, and near target site, manufacture tandem repetitive sequence and double-strand break the two, it has improved efficiency and the specificity of target-specific restructuring greatly.This TREC method enough seamless transformation mycoplasma genitalium genome effectively in yeast.
I. generate the TREC box
In another embodiment, merge by " repetition " fragment (describing at embodiment 4B (ii)) that makes (GAL1/I-SceI)-URA3 fusion product (as in embodiment 4B (i), described and produce) and 358bp be arranged in the upstream of target gene seat, generate TREC mutagenesis construct.Carry out the fusion of (GAL1/I-SceI)-URA3 product and repeated fragment by merging PCR as follows.Chimeric primers Fusl and Fus2 (being listed in top table 13) that each has with the part of (GAL1A-SceI)-URA3 fusion product and repeated fragment homology, be used in utilization (GAL1 A-SceI)-URA3 fusion product as in the pcr amplification of template and in the pcr amplification of another utilization " repetition " fragment as template.Product then carries out without primer PCR, described in embodiment 4A and 4B in the above, to generate ((GAL1/I-SceI)-URA3)-repeat fusion product.
((GAL1A-SceI)-URA3)-repeat fusion product then to use and be listed in Sce-Intl and the MUT-70 primer amplification in top table 13.As shown in that table, the homologue that each of these primers comprises ((GAL1/I-SceI)-URA3)-repeat fusion product and at 5 ' 50 base pair (bp) homologues of the terminal portions (5 ' small letter part) of target region.The Sce-Intl primer also comprises I-SceI recognition site (underscore).
Resulting TREC box (being illustrated in Figure 10 C) comprises in the following order: the 50bp homologue (downstream of the single base deletion be corrected) of the 50bp homologue (upstream of single base deletion) of 5 ' part of target region, CORE box (by gene and the URA3 mark of 18bp I-SceI recognition site, GAL1 promotor, coding I-SceI endonuclease), " repetition " (with the 358bp part of lucky target wild type gene seat upstream gene group sequence homology) and target region 3 ' part.
Therefore, once design this box in order to be transformed into yeast host cell, with these box displacement (passing through HR) 450 base pair target regions in mycoplasma genitalium genome C DS 139 locus, to in genome, produce the zone that comprises 2 tandem repetitive sequences at URA3 selective marker flank (being labeled as the large arrow of " repetition " in Figure 10 B) and endonuclease cleavage site, described cleavage site can promote the endonuclease expression of enzymes to be induced cutting by growth in semi-lactosi.In method for protrusion, comprise tandem repetitive sequence as repeated in series connection, to allow by the seamless disappearance of homologous recombination between two tumor-necrosis factor glycoproteinss.As in delitto perfetto method, comprise and induce endonuclease gene and endonuclease recognition site to allow inducing the generation double-strand break in the site of expectation restructuring.By growing comprising on the substratum of FOA, select by the seamless disappearance of restructuring.
Ii. transform and select
Use the lithium acetate integration transformation, the TREC box is introduced and comprised the genomic yeast strain of mycoplasma genitalium.Select independent Ura+ transformant and use the diagnostic primers Seq-F that lists in table 13 in the above and M2-detl (in Figure 10 C shown in little single arrow of insertion point flank), by pcr analysis, with confirm gene in the donor gene group correct position insert.Use these primer PCRs to comprise the genome that inserts the TREC box and will produce the 2.884kb product.Product from the PCR reaction separates on sepharose, and it is manifested to verify correct insertion URA3 gene.
Iii. induce endonuclease expression of enzymes, FOA to select and assessment
The clone is replica plating on the flat board that comprises SG (synthetic semi-lactosi)-His and SD-HIS (comprising glucose) then, and grows 24 hours.Grown comprising on the SG substratum of semi-lactosi as sole carbon source, to induce the expression of the I-SecI endonuclease that the GAL1 promotor controls.Under identical condition, on the SD-HIS flat board, grown, in contrast.The endonuclease expression of enzymes is intended just being positioned at the 18bp recognition sequence with the downstream in genome homology zone by cutting in box, produces double-strand break.After incubation 24 hours, induce and contrast (not inducing) cell replica plating on the SD-HIS flat board that comprises FOA (SD-HIS+FOA), to select to have lost the cell of URA3 mark, as described in embodiment 4A.
Experienced the cell that semi-lactosi induces and produced a large amount of colonies when growth on SD-HIS+FOA.On the other hand, compared with control cells, produce colony seldom.Cell rule again (restreak) come from and induce and the single colony of FOA resistance of inducing cell not with acquisition.Use Seq-F and M2-detl diagnostic primers (being listed in top table 13) to be diagnosed PCR whether to comprise with the FOA resistant cell of determining selection the genome that TREC box wherein has been removed the seamless disappearance of part that produces the target gene seat.The genomic PCR that comprises seamless disappearance produces the 0.55kb product.All 24 colonies that detect from the semi-lactosi inducing cell comprise to have wishes the mycoplasma genitalium genome of modifying.Only be separated to 2 positive colonies in the cell of never inducing.
Front 10 that induce and the clones that do not induce that the diagnosis pcr analysis detected by MPCR further analyze the genomic integrity of mycoplasma genitalium, described in embodiment 4A and 4B in the above.From the semi-lactosi of all 10 detections, induce the PCR of the DNA of colony to produce complete copy (all 10 amplicons); Do not generate whole replicon (data do not show) to from inducing cell DNA not, carrying out PCR.These results are summarised in top table 14.Result shows that part bacterium donor gene group is efficiently carried out seamless disappearance by success in yeast host cell.
Iv. example T REC
Assemble fully synthetic mycoplasma genitalium genome (approximately 583kb) and be cloned in yeast as cyclic plasmid---in yeast saccharomyces cerevisiae.The trial that comprises the anti-genetic method transformation colone genome of selecting of URA3/5-fluororotic acid (5-FOA) by standard has shown the high background 5-FOA resistance clone that comes from the bacterial genomes spontaneous deletion kept in yeast.Here, we report and a kind ofly can in yeast, efficiently accurately modify the method for bacterial genomes.The method comprises two serial homology recombination event.At first, target region is replaced with mutagenesis-cartridge, and described mutagenesis-cartridge forms with the identical DNA fragmentation of sequence with the target site upstream by knocking out CORE (l8-bp I-SceI recognition site, I-SceI gene and URA3 gene) under the GAL1 promotor is controlled.This displacement is created on the tandem repetitive sequence of CORE flank.The second, semi-lactosi abduction delivering I-SceI, it generates double-strand break (DSB) in the I-SceI site.This DSB helps homologous recombination in the cell between tumor-necrosis factor glycoproteins, causes the excision of CORE.As a result, it forms seamless modification.The method can be applicable to the range gene group modifies, and therefore provides the optional approach of modifying and design the synthetic gene group in yeast.
Materials and methods
Yeast strain and substratum
(Lartigue etc. (2009) Science, 325,1693-1696 as described earlier; With (2008) PNAS USA such as Gibson, 105,20404-20409), the Wine brewing yeast strain VL6-48N that structure comprises the full genome YAC of 0.6Mb mycoplasma genitalium (MAT α his3-Δ 200 trp1-Δ 1 ura3-52 lys2 ade2-101met14) and W303a (MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 cau1-100 RAD5).Yeast cell is growth (Amberg etc. (2005) in standard rich medium (YEPD) and synthetic glucose (SD) or synthetic semi-lactosi (SG) minimal medium Method in yeast heredity: cold spring harbor laboratory's procedures manual (Methods inyeast genetics; A Cold Spring Harbor Laboratory Course Manual), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.230).With the SD substratum that is supplemented with 5-fluororotic acid (5-FOA), the URA3 gene is lost and selected (Boeke etc. (1984) Mol Gen Genet, 197,345-346).
Produce mutagenesis-cartridge
All primer customizations synthetic (Integrated DNA Technologies).Length is greater than the primer of 60bp by the polyacrylamide gel electrophoresis purifying.Be summarised in table 13 for the primer that builds all mutagenesis-cartridges.Amplification URA3 gene from plasmid pRS306 (1,066bp) (be described in R.S.Sikorski and P.Hieter, in Genetics 122,19 (1989)); Amplification GAL1 promotor (450bp) from plasmid pYES2 (Invitrogen); From plasmid pGSKU, amplification comprises 1 of GAL1 promotor and I-SceI gene, and the 184bp fragment (Storici etc. (2003) PNAS USA., 100,14994-14999); With from plasmid pBS185 amplification Cre recombinase gene (1,032bp) (Sauer and Henderson (1990) New Biologist 2,441-449).
With Takara Ex Taq archaeal dna polymerase (Takara Bio Inc.), the condition of using manufacturers to recommend is carried out all PCR.Recombinant PCR technology by thering is slight improvements (Shevchuk etc. (2004) Nucleic Acids Res, 32, el9) carry out gene fusion.In the fusion situation of each PCR-based, the overlapping 40bp of complementary end (table 13).In order to generate each final mutagenesis-cartridge, the chimeric primers (table 13) of the 50bp homologue that fusion product comprises target site by each carries out PCR-and increases.
The structure that contains the primer of dash at tundish is chimeric; Lowercase indication mycoplasma genitalium homologous sequence; Capitalization is indicated non-homogeneous sequence; With add UnderscoreBe the I-SceI cleavage site.
Transform and pcr analysis
Carry out the Lithium Acetate integration transformation according to disclosed method (Gietz etc. (1992) Nucleic Acids Res, 20,1425).Use 2 to 3 μ g construction and integration body DNA and 25 μ g carrier DNAs (salmon testis DNA, Sigma) in normal experiment.According to disclosed scheme (Kouprina and Larionov, Nat.Protoc.3,371 (2008)), as described in embodiment 3, separated total DNA for pcr analysis from yeast in the above.Use is positioned at the primer (table 13) of target site upstream and downstream, verifies the correct integration of each mutagenesis-cartridge by PCR.Multiplex PCR is for confirming mycoplasma genitalium clone's integrity, as before (Gibson etc. (2008) PNAS USA, 105, describe in 20404-20409).(scope is from 125bp to 1025bp to produce 10 amplicons to be designed for the primer sets (organizing 3) of MPCR, with 0.1kb, increase progressively), it is around the about every 60kb DNA of the mycoplasma genitalium genome (Gibson etc. that distributed, PNAS USA, 105 (51): 20404-9 (2008)).
Result
Transform the point mutation in the MG259 locus in the synthetic mycoplasma genitalium genome kept by traditional method and relate to 2 homologous recombination events (Figure 12 A) in yeast.After the first homologous recombination, by PCR, confirm accurately to replace the target region in the synthetic gene group with the URA3 gene.But, second take turns homologous recombination after, we can not be by correctly being replaced (Figure 12 B and table 15) from 5-FOA resistance colony PCR Screening and Identification URA3 gene by DNA fragmentation.
Table 15: several cerevisiae dna modifying method are transformed the genomic efficiency of mycoplasma genitalium in yeast
Figure BDA00002358547601151
Single PCR primer sets is for analyzing FOA+ clone's correct displacement.10 primer pairs are for multiplex PCR assay.Produce all 10 amplicons and be considered to complete genome.
These results show, accurate homologous recombination does not occur between the DNA fragmentation entered and target site.The loss of URA3 mark may be due to the disappearance of not expecting.As the mycoplasma genitalium genome of ring-type YAC propagation, except Histidine prototroph, its host is not had to supplementing on function in yeast.Existence may be neutral for yeast host for any disappearance of bacterial genomes and rearrangement.Multiplex PCR is for assessing the genomic integrity of mycoplasma genitalium at yeast.The design primer sets is to produce 10 amplicons (scope, from 125bp to 1025bp, increases progressively with 0.1kb), and it distributes around the about every 60kb of mycoplasma genitalium genome.The total DNA prepared from 22 5-FOA-resistance colonies does not produce all 10 amplicons (Figure 12 C).Two amplicons have been lost in all clones, 0.525 and 0.625kb (in the mycoplasma genitalium genome contiguous).The MG259 locus is in 3kb place, the upstream of 0.65kb amplicon.In the mycoplasma genitalium genome that this result shows to breed in yeast, some spontaneous deletions or rearrangement have occurred.The loss of URA3 mark may be due to the homologous recombination between tumor-necrosis factor glycoproteins in the mycoplasma genitalium genome.As a result, the cell that has URA3 mark disappearance can be survived on the 5-FOA substratum.The probability higher (Figure 12 D) of the DNA fragmentation displacement that the likelihood ratio URA3 of the non-specific loss of URA 3 gene is entered.We also adopt two kinds of additive methods to transform identical locus and produce point mutation or the 450bp disappearance, repeat method for protrusion by delitto perfetto or series connection respectively and carry out (strategy is depicted in Figure 10 A and 10B).But we can not screen 5-FOA resistance colony by PCR, identify any correct modification (table 15).Thereby we reach a conclusion, most of 5-FOA-resistance colony comes from the cell of non-specific loss URA3 mark during operating process.
By introduce DSB near target site, the recombination frequency between two series connection repeat may improve greatly.Therefore, we are in conjunction with two strategies---and series connection repeats method for protrusion and delitto perfetto method.By merging CORE box and target site upstream 358bpDNA, produce the mutagenesis construct.To produce two tumor-necrosis factor glycoproteinss around CORE with this construct displacement 450bp target region, described CORE comprises I-SceI recognition site (Figure 13).Then, the homologous recombination between repetition will produce seamless disappearance.After conversion mutagenesis construct enters yeast, on the negative HIS agar of SG-, induce I-SceI endonuclease expression of enzymes.After 24-hour incubation, cell is replica plating on the negative HIS+5-FOA agar of SD-.On SD-HIS+5-FOA agar with the semi-lactosi inducing cell than the obvious generation of the cell of not inducing more colony (data do not show).Come from inducing cell and not the 5-FOA resistant cell of inducing cell rule again and selection and analysis list colony.Identify the transformant with correct disappearance by PCR.There is the DNA that accurately removes the CORE box and will cause generating the 0.55kb amplicon.All 24 colonies that come from the semi-lactosi inducing cell comprise the correct modification of mycoplasma genitalium genome; Only separate 2 positive colonies (data do not show) from the colony that comes from inducing cell not.Further assess the mycoplasma genitalium genomic integrity by multiplex PCR.Detected induces clone's DNA to produce whole 10 amplification subgroups from 10.Do not generate 10 whole amplification subgroups (data do not show) from the DNA of inducing cell not.Therefore, show that from the result of two pcr analysis the TREC method can carry out efficient seamless disappearance (table 15) to the bacterial genomes that is cloned into yeast.
Finally, compared aspect the disappearance of the bacterial genomes of cloning the efficiency of the efficiency of TREC method and Cre-LoxP system in yeast.The Cre-LoxP system is efficient locus specificity recombination method.It successfully in the biology of wide region for remove selective marker and large genomic DNA fragment (Gueldener etc. (2002) Nucleic Acids Res, 30, e23).Manufacture mutagenesis construct (materials and methods) by two-wheeled PCR.Two mutant LoxP sites that it is flank by URA3 mark, Cre gene under the GAL1 promotor is controlled and two end sequences of take with the target site homology form (Figure 15).Mutant LoxP site prevent reverse recombination event (Araki etc. (1997) Nucleic Acids Res, 25,868-872).
Adorned same area is by this construct target before.Carry out similar process and analyze to produce and detection site specificity disappearance.The disappearance that the 5-FOA resistance isolate of pcr analysis demonstration 93% (28/30) comprises expectation, and the multiplex PCR result shows that 100% (4/4) isolate with correct disappearance comprises complete mycoplasma genitalium genome (table 15).In sum, aspect the transformation mycoplasma genitalium genome of cloning in yeast, the efficiency of the efficiency of TREC method and Cre-LoxP system is suitable.
Seamless genome manipulation needs the anti-selective marker that can be selected and be removed subsequently usually.Several existing methods that are applicable to the anti-URA3/5-FOA of selection systems successfully shown for modifying at yeast chromosomal (Rothstein (1991) method Enzymol, 194,281-301).But we have pointed out that these methods are not suitable for transforming free ground (episomally) and remain on the mycoplasma genitalium genome in yeast.Show that synthetic mycoplasma genitalium genome stably remains in yeast, though genome comprise the tumor-necrosis factor glycoproteins that reaches 4% (Peterson etc. (1995) PNAS USA, 92,11829-11833).Therefore, in the time of in remaining on yeast, spontaneous deletion or rearrangement still can occur with low frequency.Thereby this will generate potentially the negative clone of unwanted URA3-and make 5-FOA select site-specific mutagenesis complicated during operating process.
We prove that the TREC method can generate the genomic seamless modification of mycoplasma genitalium effectively in yeast.It is a kind of simple method, only need to once transform and be applicable to the modification (insertion, gene substitution or point mutation) of other kinds.The preparation of mutagenesis construct is less than one day.In fact, do not carry out fusion reaction, we find that cotransformation CORE box and the 50bp repeated fragment overlapped each other enough obtain correct displacement (data do not show).When using the TREC method, high-frequency homologous recombination is mainly owing to the fact: in principle, during inducing DSB, each cell participates in reparation and repairs substrate (tumor-necrosis factor glycoproteins and DSB) very approaching.The performance of TREC is suitable with the Cre/LoxP system.But, because TREC does not stay trace (scar), it is more valuable for the genome manipulation aspect than Cre/LoxP system.Recently, a kind of new method, be called MIRAGE, the shown efficient seamless modification that produces Yeast genome.The method is based on repeating to introduce inverted repeat near the target site of flank take two short series connection.Inverted repeat has not promoted two excisions of connecting between repeating greatly quietly.But, due to replication slippage, inverted repeats also introduced the out of true disappearance potential problems (Gordenin etc. (1992) PNAS USA, 89,3785-3789).The another kind of shortcoming of using the MIRAGE method is to produce to knock out construct and expend time in, because its needs preparation in two days.
Conveying carry the initial cell of getting back to it as the engineering bacterial genomes of YAC can determine the function of gene and gene cluster and adjusting (Vrancic etc. (2008) Food Tech Biotechnol 46,237-251).Seamless modification is the favourable mode of transformation YAC, because the sequence outside the retention of transformation site can be given birth to unforeseeable result by potential real estate.In addition, much higher eucaryotic cells karyomit(e) comprises very most of tumor-necrosis factor glycoproteins.Their gene (one or more) that method described herein is cloned into yeast for modification is useful.We have also applied the method and generated the seamless disappearance of III type restriction enzyme in the large colony of the mycoplasma mycoides that is cloned into yeast (mycoplasma mycoides LC) genomes.Confirm accurate disappearance by order-checking.Genome subsequently shifts has manufactured the mycoplasma mycoides LC bacterial strain with genomic deletion, and it is difficult to manufacture (Lartigue etc. (2009) Science, 325:1693) due to genetic tool limited in this biology in host cell.Yeast has successfully shown that conduct is for assembling the complete genomic host of mycoplasma genitalium.TREC provides design to can be used for manufacturing the arbitrary way of the synthetic gene group of synthetic cell.
The use of yeast provides advantage than intestinal bacteria because in intestinal bacteria the clone to be greater than the foreign DNA of 300kb uncommon, it has limited its application.On the other hand, yeast cell provides the ability of megabase to the clone.
Embodiment 4D
The Cre-LoxP modification system
In order to compare, the known modification system Cre-LoxP system of modifying for bacterial genomes, for modifying identical bacterial genomes at yeast host cell.The Cre-LoxP system is known effective locus specificity recombination method, its successfully in a large amount of different biologies for removing selective marker and large genomic DNA fragment.See Gueldener etc., Nucleic Acids Res, 30, e23 (2002).
By two-wheeled PCR reaction, produce the Cre-LoxP mutagenesis construct with mutant LoxP gene, described at embodiment before.Mutant LoxP prevents reverse recombination event, as at Araki, and K. etc., Nucleic Acids Res, described in 25,868-872 (1997).
I. generate the Cre-LoxP box
Use Cre-F and Cre-R primer (table 13), from plasmid pBS185 (Sauer and Henderson.New Biologist 2,441-449 (1990)) amplification Cre recombinase gene ORF fragment (1,032bp), use primer Gal-F and Gal-R (table 13), from plasmid pYES2 (Invitrogen, Carlsbad, CA) amplification GAL1 promotor (450bp), with as described in embodiment before, produce 1066bp URA3 gene fragment by the PCR that uses Ura-F and Ura-R primer (table 13), produce the loxP-RE-GAL1-Cre-URA3-loxP-LE mutagenesis-cartridge.
Use the PCR of Cre-Fus2 and Cre-Fus4 primer (table 13) to merge generation GAL1-Cre-URA3 fusion product, and by using chimeric primers Lox-F and Lox-R (table 13) amplification fusion product, introduce mutant LoxP site, described chimeric primers comprises the part with GAL1-Cre-URA3 fusion product and mutant LoxP site homology.This amplification generates the LoxP-RE-GAL1-Cre-URA3-LoxP-LE fusion product.This fusion product is then used Int-F2 and the Int-R2 primer amplification of listing in table 13 in the above.As shown in that table, the homologue that each of these primers comprises the LoxP-RE-GAL1-Cre-URA3-LoxP-LE fusion product and 5 ' 50 base pair (bp) target region homologous fragments (lowercase type).
Gained LoxP-RE-GAL1-Cre-URA3-LoxP-LE mutagenesis-cartridge (diagram in Figure 10 D) comprises in the following order: the 50bp homologue (downstream of single base deletion) of the 50bp homologue (upstream of single base deletion) of 5 ' part of target region, a LoxP site (LoxP-RE), GAL1 promotor, Cre recombinase gene ORF, URA3 mark, the 2nd LoxP site (LoxP-LE) and target region 3 ' part.Therefore, once design this box in order to be transformed into yeast host cell, with these box displacement (passing through HR) 450 base pair target regions in mycoplasma genitalium genome C DS139 locus, to in genome, produce the zone that comprises the loxP site, can be by semi-lactosi, growth inducing is from the expression Cre of same cartridge, recombinated and lacked in this site of target inductively.Can be by growing on the FOA substratum, the disappearance of the box that selection comprises the URA3 mark.
Ii. transform and select
Use the lithium acetate integration transformation, the LoxP-Cre box is introduced and comprised the genomic yeast strain of mycoplasma genitalium.Select independent Ura+ transformant and use diagnostic primers Seq-F and M2-detl (little single arrow illustrates and lists at the insertion point flank in Figure 10 D) in table 13, by pcr analysis, with confirm gene in the donor gene group correct position insert.Comprise the genomic this PCR that inserts the LoxP-Cre box and can produce the 3.068kb product.Product from the PCR reaction separates on sepharose, and it is manifested to verify correct insertion box.
Iii. induce endonuclease expression of enzymes, FOA to select and assessment
The clone is replica plating growing 24 hours on the flat board that comprises SG (synthetic semi-lactosi)-His and SD-HIS (comprising glucose) then.Grown comprising on the SG substratum of semi-lactosi as sole carbon source, to induce the expression of the Cre recombinase that the GAL1 promotor controls.Intention is expressed recombinase and is induced restructuring in site with LoxP in box.After inducing, cell replica plating on the SD-HIS flat board that comprises FOA (SD-HIS+FOA) has been lost the cell of URA3 mark with selection, as described in embodiment 4A.
Use Seq-F and M2-detl primer (table 13), 5-FOA resistance colony is diagnosed to PCR (described at embodiment 4A-C), whether comprised with the FOA resistant cell of determining selection that wherein box is removed, the genome of the seamless disappearance of generating portion target gene seat.As at table 14 provided, result shows the disappearance that the 93% 5-FOA-resistance isolate (28/30) of testing comprises expectation.
By 4 clones by diagnosis pcr analysis test positive are carried out to MPCR, further analyze the genomic integrity of mycoplasma genitalium, described in embodiment 4A and 4B in the above.As shown in table 14 in the above, 100% (4/4) these colonies comprise all 10 amplicons, prove genomic integrity.
Come the result of study provided in comfortable embodiment 4C and 4D to show, for modify the mycoplasma genome in yeast host cell, (it is simple method to institute's TREC method that provides, available 1 conversion is carried out, and applicable disappearance, insertion, gene substitution and point mutation) efficiency, if be not more than, equal so the efficiency of the cre-LoxP modifying method known.But unlike the Cre-LoxP method, the TREC method produces seamless modification, makes it especially have superiority.
Embodiment 5
The transfer donator genome enters host cell, modify and shift by TREC in host cell and enter recipient cell
This embodiment has described for the transfer donator genome, entering host cell, at host cell, modify the combination that donor gene group (TREC modifications) and transfer donator genome enter the method for recipient cell of providing has been provided, and operates the donor gene group.The method is for non-existent mycoplasma mycoides LC genome in laboratory or naturally before yeast successfully transform.As following, described, from be cloned into the donor mycoplasma mycoides LC genome of yeast host cell, disappearance III type restriction enzyme allele.Select III type restriction enzyme allele, because expect it for existence and shift optional.The genome of modifying then is transferred to the mycoplasma capri recipient cell, produces the complete genomic new cell that comprises modification.
Embodiment 5A
The transfer donator genome enters host modifying factor group
Mycoplasma mycoides LC-YCp genome is transferred to yeast strain W303a propagation therein, described in embodiment 1A in the above.
In one embodiment, use the TREC method of describing in embodiment 4C in the above, with the box displacement III type restriction enzyme allele that comprises the URA3 mark, the URA3 mark is removed by 5-fluororotic acid (5-FOA) selection subsequently.This process for schematic illustrations in Figure 11, by as PCR described in above-described embodiment 4C merge and generate TREC and knock out box, details is as follows.At first generate CORE box (comprising in order GAL1 gene, SceI gene and URA3 mark).
Also by PCR, generate tandem repetitive sequence (TRS) fragment.This fragment comprises the homologue with the genomic part of mycoplasma mycoides LC of III type restriction enzyme target gene seat upstream.The TRS fragment and in the genome of target region upstream corresponding homology part in Figure 11 with large horizontal arrow leader notes.Comprise the TRS fragment, in order to exist series connection to repeat in genome after by homologous recombination, box being integrated into to genome, outstanding to promote box by restructuring.
The CORE box is by fusion PCR and the TRS segment composition described in embodiment 4 in the above.Merging primer is then using the CORE box to use as the PCR of template with in using TRS fragment another PCR as template.Product is followed combined and is carried out without primer PCR to connect product.The gained fusion product is then used increased other comprising with CORE-TRS fusion product homology with the primer in the 50bp zone of target region homology.This primer also comprises 18bp I-SceI recognition site.Therefore, TREC knocks out box and comprises in the following order: the 50bp homologue of 5 ' part of target region, 18bp I-SceI recognition site, CORE box (by this order by: gene and the URA3 mark of GAL1 promotor, the I-SceI endonuclease of encoding form), the TRS repeated fragment (with just in the part of the genome sequence homology of target gene seat upstream; In Figure 10 A, with large horizontal chuck, indicate) and target region 3 ' 50bp homologue partly.
In order to modify mycoplasma mycoides LC-YCp donor gene group in yeast W303a host, use the lithium acetate integration transformation, TREC is knocked out to box and be transformed into host cell, described in embodiment 4C (ii) in the above.In order to select to knock out box displacement TypeR III ORF (target gene seat) (by the zone of the end at box and target site 50-bp homology) with TREC, grown cell, select independent URA+ transformant and analyze.This process produces the genome that wherein III type restriction enzyme allele has been replaced by box (being labeled as typeIIIres::URA3 in Figure 11).
Cell is then grown on the flat board that comprises the SG-His substratum, so that semi-lactosi is unique carbon source.This step is induced the expression that is subject to the I-SceI endonuclease under the control of GAL1 promotor, to promote cutting 18-bpI-SceI site (asterisk in Figure 11), manufactures double-strand break.Double-strand break then can promote the homologous recombination between tandem repetitive sequence (the large arrow of level), by double-strand break, promotes.
In order to select this recombination event, cell grows to select to lose the cell of URA3 mark on SD-HIS 5-FOA substratum, and it is inferred lose the TREC box.Carry out this process to select wherein the genome (being labeled as Δ typelllR in Figure 11) of seamless disappearance typeIIIres gene.
In another kind of embodiment, with 3 steps, build and knock out box.The first, by use primer RCO293 (
Figure BDA00002358547601211
Figure BDA00002358547601212
TAGGGATAACAGGGTAATACGGAT; SEQ ID NO:152) and primer RCO294 (ATCTTGTCTATTTAATTCTAAAACAGGGTAATAACTATATAATTAA
Figure BDA00002358547601213
ATTGAAG:SEQ ID NO:153) PCR, produce and be known as the 2.3kb DNA fragmentation that knocks out core (KOC).Gained PCR product comprises gene and the URA3 mark that returns endonuclease with 50bp fragment (outstanding with black matrix in primer RCO293), 18bp I-SceI recognition site, GAL1 promotor, the coding I-SceI of 5 ' target site upstream homology since 5 ' end.The second, by primer RCO295 (AATTTAATTATATCAGTTATTACCCTGTTTTAGAATTAAATAGACA
Figure BDA00002358547601215
Figure BDA00002358547601216
AGATAATGG; SEQ ID NO:154) and primer RCO296 (A
Figure BDA00002358547601217
Figure BDA00002358547601218
GCACTACAAATTCTTGTTTATTAGTTA; SEQ ID NO:155), use the 400-bp of mycoplasma mycoides LC genomic dna as template amplification target site upstream.The PCR product that is known as tandem repetitive sequence (TRS) comprises the 50bp fragment (outstanding with black matrix in primer RCO296) with 3 ' target site downstream homology.The 3rd, two PCR product KOC of the 50bp that overlaps each other and TRS (underlining in primer RCO294 and RCO295), link together by the fusion method of PCR-based.Fusion product---knocks out box---by the gel extraction kit gel-purifying from Qiagen, and increases by primer RCO293 and primer RCO296 again.Final 2.7kb fragment is then used as (Gietz etc., Nucleic Acids Res 20,1425 (Mar 25,1992) lithium acetate (LioAc) method of) describing, be transformed into and comprise the genomic yeast 303a of mycoplasma mycoides LC bacterial strain (Benders etc., Science, submitted (2009)), and select uridylic and Histidine prototroph the two.As (Kouprina and Larionov, Nat Protoc 3,371 (2008)) describes, the total DNA of preparation from transformant.Be positioned at the primer 5 (GATTTTTATGCTGGATCTGGAACA of target site upstream by use; And be in the primer 6 (TCCGTATTACCCTGTTATCCCTA that knock out in box SEQ ID NO:192); SEQ ID NO:193) PCR screening, confirm to knock out box with the displacement of III type RE locus.In order to manufacture the unmarked disappearance of III type RE, the PCR positive strain is grown comprising on the substratum of semi-lactosi as sole carbon source, and then 5-FOA is counter selects, described at embodiment 4.Above-mentioned all pcr amplification experiments are used Phusion archaeal dna polymerase (New England Biolabs) to carry out.Use primer 5 and the primer 7 (CTACTTCAAATAGTATTCTTTTAAGCG that are positioned at the target site downstream; SEQ ID NO:194), the mycoplasma mycoides LC genome from the purifying of transformant by pcr amplification.By test kit (Qiagen) purified pcr product and for using primer 5 and 7 order-checkings.
Therefore, the design modifying method to produce the mycoplasma mycoides LC genome of two modifications in yeast host cell.The first genome of modifying be insert the TREC box after but the genome obtained before selecting in the restructuring promoted by the enzymic digestion of I-secI endonuclease with on 5-FOA.This first genome comprises (TREC) box that comprises URA3, and it is at III type restriction enzyme allele seat displacement wild type gene (Δ typeIIIres::URA).The second genome is to remove the final product obtained after box, its seamless disappearance that comprises III type restriction enzyme allele (Δ typeIIIres).
In order to show modifying factor group (the Δ typeIIIres::URA produced in this research, Δ typeIIIres) be correct size, the genomic dna separated is following electrophoresis on the CHEF gel, to compare their size and the genomic size of unmodified mycoplasma mycoides LC.For this process, rinsing yeast inserted block also spends the night with 50 AsiSI of unit, RsrII and FseI restriction enzyme (its specificity cutting pastoris genomic dna as mentioned above) digestion.The DNA inserted block then is loaded onto in 1% TAE sepharose with the pastoris genomic dna fragment electrophoresis by making digestion to outside purifying donor dna.Inserted block shifts out and residual PspXI restriction enzyme digestion for genomic dna from hole, and it makes the linearizing of mycoplasma mycoides LC genomic dna.After this digestion, all inserted blocks of rinsing application of sample are to pulsed field gel.SYBR Gold for gel (dilution 1:10,000) dyeing.With after GE Typhoon 9410 imager scanning gels, observing PFGE pattern (data do not show).
Design has the sample of Δ typeIIIres::URA and Δ typeIIIres modifying factor group and correctly shows to have the genome quite big or small with the unmodified genome.This process has produced another clone (Δ 500kb) during further being presented at research process.The size of the band based on reclaiming from this clone, its genome comprises the 500kb disappearance.This is cloned in the research of back with comparing, because infer that it lacks many indispensable genes and still retains YCp (yeast centromeric plasmid) element and tetM selective marker.
Generate another embodiment diagram in Figure 19 of III type restriction enzyme disappearance.By manufacture seamless disappearance in nonessential III type restriction endonuclease gene, transform YCpMmycl.l in yeast.Simply, the YCpMmycl.l yeast clone is at first with comprising the URA3 mark and transformed by the box of the SCEI endonuclease gene of GAL1 promotor control.Box inserts III type gene as choice criteria; 4 of 5 clones comprise complete genome, and 1 comprise there is large disappearance genome (YCpMmycl.l-Δ 500kb) (Figure 11).By the cutting of the I-SceI recognition site near box one end, remove URA3 box (Figure 19).With the anti-clone who selects generation to lose the URA3 box of 5-fluororotic acid (5-FOA).Therefore, obtain two mycoplasma mycoides YCp genomes: one comprises URA3 box and another seamless disappearance (Figure 19) that comprises III type restriction enzyme allele.Verify genomic this variation (data do not show) by PCR.
Embodiment 5B
The genome of modification is transferred to donorcells
Use the method for describing in embodiment 3 in the above, the genome that these mycoplasma mycoideses LC is modified (comprising Δ 500kb contrast) and the mycoplasma mycoides LC genome of unmodified are transferred to the mycoplasma capri recipient cell.Use in embodiment 3 in the above, describe, be illustrated in third party's case of indicating by numeral " 3 " in Fig. 8 and shifted.Described at embodiment 3, the method, before shift reaction, comprises the step that methylates, Deproteinization step (processing with Proteinase K) and melts step.Transfer is to enter the wild-type receptor cell.For this process, according to what describe, prepare the agarose inserted block in embodiment 3A (i), and with restriction enzyme mixture and the two cleaning of gel electrophoresis, as (also referring to embodiment 3A (i)) described in embodiment 5A in the above.Described at embodiment 3A (ii) and 3A (iii), existing in the 5%PEG situation, shifted, with acellular mycoplasma mycoides LC extract, methylate and protease K digesting.Described in embodiment 3 in the above, genome is transferred to wild-type (non-RE-defect) mycoplasma capri cell.By under 37 ℃, select the growth of blue colony on the SP4 substratum that comprises tsiklomitsin, assess successfully and shift.Result provides in the following Table 17.
Table 17 shifts the mycoplasma mycoides LC genome of modifying and enters wild-type mycoplasma capri recipient cell
Figure BDA00002358547601231
The quantity that shifts body represents the average of at least 3 researchs.The error of report is average deviation.
As be displayed in Table 17, it is similar to viewed quantity (the colony average out to 28 of each inserted block) when shifting the mycoplasma mycoides LC genome of unmodified that the transfer of the genome (Δ typeIIIres::URA3 and Δ typeIIIres) of two intention modifications produces the quantity (colony of each inserted block on average is respectively 28 and 33) of the blue colony of tsiklomitsin-resistance.According to expectation, shift comprise the 500kb disappearance (and infer lack essential mycoplasma base because of but reservation YCp element and tetM) contrast clone and enter the mycoplasma capri recipient cell and do not produce colony.
In order to verify the selected sequence that comprises the colony that shifts the modifying factor group, the III type locus in these genomes is checked order.Sequencing result shows, all has the modification of expection in the bacterial strain (Δ typeIIIres::URA3 and Δ typeIIIres) of two kinds of modifications.For example, the disappearance of checking typeIIIres gene in Δ typeIIIres genome, it has the typeIIImod gene that comprises typeIIIres gene in contiguous wild type gene group and is connected the nucleotide sequence of (seeing Figure 11) with the DNA in natural typeIIIres gene downstream, and this proves that gene seamless disappearance in these cells.
In order to verify that the genotype that reclaims colony is mycoplasma mycoides LC, the genomic dna by the southern blotting technique analysis from selected blue colony, it uses the IS1296 element as probe.The copy of IS1296 insertion sequence is dispersed in whole mycoplasma mycoides LC (donor) genome, but is not present in mycoplasma capri (acceptor) genome.In order to verify whole loss of III type restriction enzyme sequence in the genome of modifying, further with the probe that comprises mycoplasma mycoides LC typeIIIres gene order, survey trace.Carry out as follows southern blotting technique.
Shift to have the genomic mycoplasma capri recipient cell of mycoplasma mycoides modification or unmodified bunch from 10ml and extract the total DNA of mycoplasma.From natural mycoplasma mycoides LC clone the genomic dna of 1.1 donorcellses and from the genomic dna of mycoplasma capri recipient cell with comparing.Use Wizard genomic dna purification kit (Promega) to be extracted.For southern blotting technique hybridization, with HindIII or EcoRV, digest 1.5 μ gDNA and gained sample by electrophoresis on 1% sepharose separately.Shift (alkali transfer) by alkali and will transfer to from the DNA fragmentation of gel the nylon film (Nytran Super Charge, Schleicher and Schuell) of positively charged.By the DNA probe of 20 (20) ng/ml digoxigenin marks (IS 1296 insertion sequences and typeIIIres gene order) and film hybridization, to verify respectively mycoplasma mycoides genotype and donor gene group, modify.
The film Fab fragment incubation of the anti-digoxigenin antibody be connected with alkaline phosphatase.Then with fluorogenic substrate HNPP (2-hydroxyl-3-naphthoic acid-2 '-phenylaniline (phenylanilide) phosphoric acid salt) (Roche Molecular Biochemicals), detect hybridization.Under UV, with photographic camera with for obtaining Quantity One software (Bio-Rad Laboratories, Inc.) the detection chemiluminescence of graphical design.
Each of selected transfer colony produces identical pattern (8 band) in the trace (data do not show) of surveying with the IS1296 probe.As expected, the IS1296 pattern do not detected in comprising from the swimming lane of recipient cell (r).This result shows strongly, the LC of the mycoplasma mycoides really genotype of the colony of recovery.In addition, the swimming lane comprised from the control sample of mycoplasma mycoides donor gene group comprises the band of being identified by the typeIIIres probe.But, comprise from the swimming lane that shifts the genome sample of modifying, this band do not detected.
These results show, it is different types of recipient cell kind that the donor gene group of modifying in yeast host cell successfully is transferred to donor.Result further shows that the genome of two modifications all comprises the modification of intention (losing the TypeIIIres gene).Therefore, the method provided is successfully used to generate two kinds of synthesis of receptor cells, and it comprises the synthetic donor mycoplasma mycoides LC genome of non-existent transfer in laboratory or nature before.These results have shown in the yeast full genome of genetic manipulation bacterium and it have been installed to enter recipient cell to produce first example of new bacteria.
Embodiment 6
Donor gene group or the semisynthetic donor gene group of the chemosynthesis of assembling in yeast host cell and propagation are transferred to recipient cell
Chemosynthesis 1.08Mbp mycoplasma mycoides genome, and as the kinetochore plasmid, assemble in yeast; Genome is separated into naked DNA and is transferred to mycoplasma capri, to produce, only is synthesized the new bacterial cell that genome is controlled.
Described herein is to design, synthesize and assemble 1,077,947-bp mycoplasma mycoides JCVI-syn1 genome from 1,0781-kb synthetic DNA box.Promote assembling by the in vitro and in vivo assembly method.Recombinate and assemble the box of 10 groups and breed in yeast/shuttle vehicle by yeast.The 10-kb assembly recombinates to produce the 100-kb assembly with 10 groups.In single final step, 11 100-kb assemblies of gained are reassembled into to the complete genome group.Select and confirm to carry the yeast clone of synthetic gene group by multiplex PCR and restriction analysis.
The synthetic gene group of assembling also successfully shifts and enters restricted type feminine gender (restriction-minus) mycoplasma capri cell as kinetochore plasmid propagation in yeast.Desired phenotype character that new cell has the synthetic DNA sequence of mycoplasma mycoides and design---genetically deficient and polymorphism of comprising watermark sequence and other designs---.We claim that this bacterial strain is mycoplasma mycoides JCVI-syn1.This is the second bacterial chromosome synthesized, and surpasses 100 ten thousand bp first.It is successfully to be transferred to recipient cell and to produce the new synthetic bacterial genomes that only is synthesized the cell that karyomit(e) controls.This new synthetic karyomit(e) cell can continue self-replacation.Ability like this is confirmed in this research: start from digitized genetic information, synthetic new DNA and synthetic DNA is shifted and enters cell and replace all existing genetic information, and as a result of produce the cell of only being controlled by DNA of this synthetic design.Existing (endogenous) genetic information is lost and is as a result of produced new cell, and its synthetic karyomit(e) only be designed is controlled.
Embodiment 6A
Synthetic donor gene group design
Genome sequence (Benders etc., Nucleic Acids Res, (2010) that the height of the genomic design of mycoplasma mycoides JCVI-syn1 based on two mycoplasma mycoides subspecies capri GM12 laboratory strains of describing before accurately completes; Lartigue etc., Science 325,1693 (2009)).One is the Genome donor [GenBank accession number CP001621] (Lartigue etc., Science 317,632 (2007)) that Lartigue etc. is used.Another is by shifting the bacterial strain [GenBank accession number CP001668] (Lartigue etc., Science 325,1693 (2009)) that the genome YCpMmyc1.1-Δ typeIIIres of clone and transformation produces in yeast.Identified the difference in 95 sites between the mycoplasma mycoides genome sequence.The genome sequence (CP001668) that our choice for use successfully shifts from yeast is as our design reference; Proofread and correct as all differences between the before synthetic box with biology significance with coupling CP001668.Our the synthetic box and the sequence difference between CP001668 that appear at 19 sites seem harmless, so be not corrected.These genomes synthetic at us (JCVI-syn1) and we have cloned and have been used as the natural gene group of genome transfer criteria from yeast in yeast, 19 polymorphism difference (Lartigue etc. are provided between YCpMmyc1.1, Science 325,1693 (2009)).
I. Genome sequence
GenBank accession number CP001621 is the sequence as the mycoplasma mycoides bacterial strain of Genome donor, and as descriptions (Science 317,632 (2007)) such as Lartigue, length is 1,089,202bp.
MmycDRAFT-starts our synthetic mycoplasma mycoides genome design, and it uses from draft (draft) genome sequence that produces top sequence (CP001621) scheme, and length is 1,114,292bp.Find that this draft comprises large repetition.
GenBank accession number CP001668 is by the genetic modification that lacks Type III limiting acid endo enzyme, the sequence (Lartigue etc. that then shift the mycoplasma mycoides bacterial strain to produce mycoplasma in yeast, Science325,1693 (2009)).This sequence length is 1,084,586bp.
At first, the mmycDRAFT sequence is divided into to the box that length is 1080bp, 80bp is overlapping, and adds the NotI restriction site at each end, as follows:
Mmyc0=base 1-1080 is at front and then GCGGCCGC
Mmyc1=base 1001-2080 is at front and then GCGGCCGC
Mmyc2=base 2001-3080 is at front and then GCGGCCGC
Mmyc1113=base 1113001-1114080 is at front and then GCGGCCGC
Mmyc1114=base 1114001-1114292 is at front and then GCGGCCGC
Be arranged in the mmycDRAFT Sequence design box by Blue Heron customization for the synthesis of.Initially 1,072 box of customization comprises the box of 1,115 all designs, except mmyc0-mmyc14 (15 boxes), mmyc835-mmyc852 (18 boxes) and mmyc1105-mmyc1114 (10 boxes).
Assemble these 1,072 box and will produce the extension fragment (contiguous stretch) of two vicinities of DNA, two breach are corresponding to the uncertain zone of sequence.
Because known this genome stably remains in yeast and transferable to produce the mycoplasma that can survive, so, after it completes fully, based on sequence C P001668, design remaining box from yeast.At first, the design box is to fill the breach of two maximums:
Fill the box of breach 1." a " indication is not the box from the part of the initial customization of Blue Heron.Box 835a-850a produces coupling CP001668 and fills the sequence of the breach that box mmyc835-mmyc852 stays, its careless omission from initial Blue Heron customization.
Fill the box of breach 2.The breach that in box 2a-12ax filling draft sequence, box mmyc1105-mmyc1114 and place, mmyc0-mmyc14 place stay (these careless omissions from Blue Heron customization).This sequence exact matching CP001668 is also obviously shorter than the corresponding zone of draft sequence, and this is mainly the problem due to this sequence of assembling.The sequence of mmyc12ax is longer than major part; Therefore, " x " representative extends.
Make the synthetic gene group mate the box of CP001668 near replication origin.Much difference is arranged between mmycDRAFT sequence and CP001668 in this zone.
Mmyc799a (the overlapping mmyc798 of 80bp---to the end of 1084586, CP001668)
Mmyc811a (CP001668-80 to 1074)
Mmyc812a (CP001668 995 to 2074)
The synthetic box of coupling CP001668.
Mmyc56a (CP001668 344335 to 345333), mmyc58a (CP001668 345252 to 345926), and the mmyc938.1a between mmyc938 and mmyc939.
Box by oligonucleotide mutagenesis " fixing (fixed) " with coupling CP001668.
" f " box has sequence difference in a small amount, and it can be fixing with exact matching CP001668 by oligonucleotide mutagenesis.The details of how to fix these boxes see below.Fixing following box: mmyc1011f, mmyc1028f, mmyc247f, mmyc248f, mmyc342f, mmyc399f, mmyc400f, mmyc528f, mmyc529f, mmyc578f, mmyc579f, mmyc632f, mmyc642f, mmyc759f and mmyc874f.In addition, in order to build, have " 94D " disappearance genome---it lacks mmyc936-mmyc939, produce box mmyc940f overlapping to comprise with 5 of box mmyc935 '.By it is lacked from the natural gene group of yeast, then shift the mycoplasma that can survive to produce, show that this zone is dispensable.
The box that comprises watermark sequence.
(1) (5 ' 80bp is overlapping from mmyc282 for mmycWM1 displacement mmyc282-287; 3 ' 80bp is overlapping from mmyc287), (2) mmycWM2b displacement mmyc447 (two 80bp are overlapping from mmyc447), (3) mmycWM3 displacement mmyc106 (two 80bp are overlapping from mmyc106), and (4) mmycWM4 displacement mmyc680 (two 80bp are overlapping from mmyc680).
Nucleotide 1 is at the initiator codon place of dnaA gene.
Two kinds of mycoplasma mycoides sequence C P001621 and CP001668 are used coding scheme, and wherein Nucleotide 1 is at the initiator codon place of dnaA gene, near the DNA replication dna initial point.But the mmycDRAFT sequence designed for initial box is from different initial points with identical direction encoding.Therefore, the Nucleotide 1 of genome sequence is arranged in box mmyc811a.
Difference between loose box design and CP001668.
Between box design and CP001668 sequence, have 19 to seem harmless difference, we select not fix them.These are all the difference of homopolymer or dinucleotides test length, and between the gene of annotation.Some may affect the interval between promotor-35 and-10 sequences, and therefore can affect genetic expression.Table 18 has provided the details of these 19 differences.Except watermark sequence, they also provide more sequence difference so that synthetic gene group and the natural mycoplasma mycoides genome of having cloned in yeast are distinguished.
DNA box sequence is proofreaied and correct
Based on incomplete draft sequence, customization comprises the synthetic box of synthetic gene group.Because this has little difference between the synthetic box sequence customized and complete base mycoplasma mycoides subspecies capri genome sequence.43 boxes comprise difference.Much difference is not predict little insertion or the disappearance that is arranged in gene in homopolymer or dinucleotides test.These variations are thought optimum and fixing.Select 16 boxes to be fixed.In 1 box, 12bp repeating unit disappearance, difference is all that single base changes.
Adopt 2 strategies to carry out the mycoplasma mycoides subspecies capri genome sequence of matching complete to change synthetic box sequence.Quik
Figure BDA00002358547601271
The site-directed mutagenesis kit of II (Stratagene) changes for some single bases.Remaining variation realizes (Gibson etc., NatMethods 6,343 (2009)) by the combination of PCR and vitro recombination.The plasmid skeleton that primer (BH pUC bckbn For1 and Rev1) is used for the recombining reaction that increases.In conjunction with correct errors primer (box Fix For1 and Rev1), use the complement of skeleton primer (BH inserts For1 and Rev1) to there is amplicon and the BH skeleton amplicon in the zone of homology each other with generation.3 kinds of boxes (data do not show) that the PCR product is proofreaied and correct with generation for vitro recombination.All stuck-at-１s, 080bp box clone is upper to implement.
Primer BH pUC bckbn For1
(5 '-CGTCAAAGCAACCATAGTACGCGCCCTGTAG-3 '; SEQ ID NO.:#) and BHpUC bckbn Rev1 (5 '-CTGACTCGCTGCGCTCGGTCGTTCGGC-3 '; The plasmid skeleton of SEQ ID NO.:#) cloning for the box that increases.This amplicon (BH skeleton) is ~ 2, and 600bp and comprise amicillin resistance mark and replication origin.Primer BH inset For1 (5 '-GCCGAACGACCGAGCGCAGCGAGTCAG-3 '; SEQ ID NO.:#) and BH inset Rev1 (5 '-CTACAGGGCGCGTACTATGGTTGCTTTGACG-3 '; SEQ ID NO.:#) be the reverse complement (reverse complement) of BHpUC bckbn For1 and BH pUC bckbn Rev1, and as thering is the forward of proofreading and correct primer and reverse primer with amplification box inset and producing the homology zone with BH skeleton for vitro recombination.
For a change the single core thuja acid in box, need two oligonucleotide.The first primer comprises the fixing nucleotide sequence that flank is an about 20-25 base, and the second primer is the complement of the first primer.Shown for Nucleotide being changed over to the example of the primer of G.Box Fix For1
(5 '-GAACTGAAAATTATATATCAGGTA
Figure BDA00002358547601272
ATATGAATAGGAAATAGTATGTC-3 ' SEQ ID NO.:#) and box Fix Rev1
(5 '-GACATACTATTTCCTATTCATAT
Figure BDA00002358547601273
TACCTGATATATAATTTTCAGTTC-3 ' SEQ ID NO.:#), treat that reformed Nucleotide is runic.Use PCR to change selected base.Primer BH inset For1, and use inset part that box Fix Rev1 amplification proofreaies and correct and primer BH inset Rev1 and box Fix For1 also to there is the residue inset of correction for amplification.Because box Fix For1 and box Fix Rev1 primer are reverse complements each other, this produces the homology zone, for the vitro recombination between amplicon.As previously mentioned, BH inset primer produces the homologue with HB skeleton fragment, and this allows the vitro recombination reaction of 3 fragments, to produce the inset plasmid of proofreading and correct.
A repetition that synthetic box comprises five 12bp sequences, and complete genome sequence shows six repetitions of this sequence.PCR and vitro recombination are as above used to proofread and correct the 12bp disappearance with minimum change.4 repeating units that comprise 5 ' end for the forward primer that adds the 12bp repeating unit are followed 27 bases of non repetitive sequence on 3 ' end.This primer is used to be created in the amplicon that 5 ' end has 4 repeating units together with primer BH inset Rev1.The complement that comprises 5 ' hold 4 repeating units for the reverse primer that adds the 12bp repeating unit is 27 bases of non repetitive sequence on 3 ' end then.This primer is used to be created in the amplicon that 3 ' end has 4 repeating units together with primer BH inset For1.After vitro recombination, the clone who recombinates between order-checking clone two unit with two repeating units and 5 of confirming amplicon 3 ' end ' hold, its generation has the box inset sequence of 6 12bp repeating units.
In box 940, carried out and synthetic and natural sequence between the incoherent further change of difference.Box 936-939 is difficult to assembling.Further work shows that box 936-939 not necessarily also can be lacked for the existence of mycoplasma mycoides subspecies capri.For this zone of disappearance during structure synthetic gene group, the overlapping 80bp with matching box 935 3 ' of 5 ' 80bp that changes box 940 is overlapping.Once build, box 935 can then combine with the box 940 of disappearance box 936-939.For a change overlapping region, use BH inset Rev1 and the forward primer amplification box 940 that is close to 5 ' 80bp overlapping region combination.By obtaining from 5 of box 936 ' overlapping (its 3 ' 80bp with box 935 is overlapping identical) with BH inset For1 and reverse primer amplification box 936, described reverse primer is overlapping in conjunction with 3 ' 40bp's---and it has for 5 of the complement of the forward primer for the box 940 that increases ' extension.Vitro recombination is then used together with BH skeleton fragment, with generation, has the box 940 overlapping with 5 ' 80bp of box 935.
Ii. Watermark
For further distinguishing synthetic gene group and natural gene group, designed 4 watermark sequences to replace one or more boxes, be added on the other sequence of insertion and will can not disturb the place of viability.
Watermark-1,321 a uncoded word symbol, 1246 base pairs
J.CRAIG?VENTER?INSTITUTE?2009
ABCDEFGHIJKLMNOPQRSTUVWXYZ
0123456789#@()-+\=/：<；>$&}{*]″[％!′.，
SYNTHETIC?GENOMICS，INC.
<!DOCTYPE?HTML><HTML><HEAD><TITLE>GENOME?TEAM</TITLE></HEAD><BODY><A?HREF="HTTP：//WWW.JCVI.ORG/″>THE?JCVI</A><P>PROVE?YOU′VE?DECODED?THIS?WATERMARK?BY?EMAILING?US<A
HREF=″MAILTO：MROQSTIZ@JCVI.ORG″>HERE!</A></P></BODY></HTML>
Figure BDA00002358547601281
TTAACTAGCTAAGTTCGAATATTTCTATAGCTGTACATATTGTAATGCTGATAACTAATACTGTGCGCTTGACTGTGATCCTGATAAATAACTTCTTCTGTAGGGTAGAGTTTTATTTAAGGCTACTACTGGTTGCAAACCAATGCCGTACATTACTAGCTTGATCCTTGGTCGGTCATTGGGGGATATCTCTTACTAATAGAGCGGCCTATCGCGTATTCTCGCCGGACCCCCCTCTCCCACACCAGCGGTGTACATCACCAAGAAAATGAGGGGAACGGATGAGGAACGAGTGGGGGCTCATTGCTGATCATAATGACTGTTTATATACTAATGCCGTCAACTGTTTGCTGTGATACTGTGCTTTCGAGGGCGGGAGATTCTTTTTGACATACATAAATATCATGACAAAACAGCCGGTCATGACAAAACAGCCGGTCATAATAGATTAGCCGGTGACTGTGAAACTAAAGCTACTAATGCCGTCAATAAATATGATAATAGCAACGGCCTGACTGTGAAACTAAAGCCGGCACTCATAATAGATTAGCCGGAGTCGTATTCATAGCCGGTAGATATCACTATAAGGCCCAGGATCATGATGAACACAGCACCACGTCGTCGTCCGAGTTTTTTTGCGCGACGTCTATACCACGGAAGCTGATCATAAATAGTTTTTTTGCTGCGGCACTAGAGCCGGACAAGCACACTACGTTTGTAAATACATCGTTCCGAATTGTAAATAATTTAATTTCGTATTTAAATTAATGATCACTGGCTATAGTCTAGTGATAACTACAATAGCTAGCAATAAGTCATATATAACAATAGCTGAACCTGTGCTACATATCCGCTATACGGTAGATATCACTATAAGGCCCAGGACAATAGCTGACTGACGTCAGCAACTACGTTTAGCTTGACTGTGGTCGGTTTTTTTGCTGCGACGTCTATACGGAAGCTCATAACTATAAGAGCGGCACTAGAGCCGGCACACAAGCCGGCACAGTCGTATTCATAGCCGCACTCATGACAAAACAGC
Figure BDA00002358547601291
(SEQ?ID?NO:#)
Watermark-2326 a uncoded word symbol, 1081 base pairs
MIKKEL?ALGIRE，MICHAEL?MONTAGUE，SANJAY?VASHEE，CAROLELARTIGUE，CHUCK?MERRYMAN，NINA?ALPEROVICH，NACYRAASSAD-GARCIA，GWYN?BENDERS，RAY-YUAN?CHUANG，EVGENIADENISOVA，DANIEL?GIBSON，JOHN?GLASS，ZHI-QING?QI。
"TO?LIVE，TO?ERR，TO?FALL，TO?TRIUMPH，TO?RECREATE?LIFE?OUT?OF?LIFE."-JAMES?JOYCE
Figure BDA00002358547601292
TTAACTAGCTAACAACTGGCAGCATAAAACATATAGAACTACCTGCTATAAGTGATACAACTGTTTTCATAGTAAAACATACAACGTTGCTGATAGTACTCCTAAGTGATAGCTTAGTGCGTTTAGCTATATTGTAGGCTTCATAATAAGTGATATTTTAGCTACGTAACTAAATAAACTAGCTATGACTGTACTCCTAAGTGATATTTTCATCCTTTGCAATACAATAACTACTACATCAATAGTGCGTGATATCCTGTGCTAGATATAGAACACATAACTACGTTTGCTGTTTTCAGTGATATGCTAGTTTCATCTATAGATATAGGCTGCTTAGATTCCCTACTAGCTATTTCTGTAGGTGATATACGTCCATTGCATAATTAATGCATTTAACTAGCTGTGATACTATAGCATCCCCATTCCTAGTGCATATTTTCATCCTAGTGCTACGTGATATAATTGTACTAATGCCTGTAGATAATTTAATGCCTGGCTCGTTTGTAGGTGAAATTTAGTGCCTGTAAAACATATACCTGAGTGCTCGTTGCGTGATAGTTCGTTCATGCATATACAACTAGGCTGCTGTGATATGGTCACTGCCCTTACTGTGCTACATATTACTGCGAGGGGGATGACTATAAACCTGTTGTAAGTGATATGACGTATATAACTACTAGTGATATGACGTATAGGCTAGAACAACGTGATATGACGTATATGACTACTGTCCCAAACATCAGTGATATGACGTATACTATAATTTCATAATAGTGATAAATAAACCTGGGCTAAATACGTTCCTGAATACGTGGCATAAACCTGGGCTAACGAGGAATACCCATAGTTTAGCAATAAGCTATAGTTCGTCATTTTTAA
Figure BDA00002358547601293
(SEQ?ID?NO:#)
Watermark-3335 a uncoded word symbol, 1109 base pairs
CLYDE?HUTCHISON，ADRIANA?JIGA，RADHA?KRISHNAKUMAR，JAN?MOY，MONZIA?MOODIE，MARVIN?FRAZIER，HOLLY?BADEN-TILSON，JASONMITCHELL，DANA?BUSAM，JUSTIN?JOHNSON，LAKSHMI?DEVIVISWANATHAN，JESSICA?HOSTETLER，ROBERT?FRIEDMAN，VLADIMIRNOSKOV，JAYSHREE?ZAVERI。
"SEE?THINGS?NOT?AS?THEY?ARE，BUT?AS?THEY?MIGHT?BE."
Figure BDA00002358547601294
TTAACTAGCTAATTTAACCATATTTAAATATCATCCTGATTTTCACTGGCTCGTTGCGTGATATAGATTCTACTGTAGTGCTAGATAGTTCTGTACTAGGTGATACTATAGATTTCATAGATAGCACACTGGCTTCATGCTAGGCATCCCAATAGCTAGTGATAGTTTAGTGCATACAACGTCATGTGATACAACGTTGCTGGCTGTAGATACAACGTCGTATTCTGTAAGTGATACAATAGCTATTGCTGTGCAAGGCCTATAGTGGCTGTAACTAGTGATATCACGTAACAACCATATAAGTTAGATTTAATGCCCCTGACTGAACGCTCGTTGCGTGATAGTTTAGGCTCGTTGCATACAACTGTGATTTTCATAAAACACGTGATAATTTAGTGCTAGATAAGTTCCGCTTAGCAAGTGATAGTTTCCGCTTGACTGTGCATAGTTCGTTCATGCGCTCGTTGCGTGATAAACTAGGCAGCTTCACAACTGATAATTTAATTGCTGAATTGCTGGCTGTCTAGTGCTAGTGATCATAGTGCGTGATAGTTTAAGCTGCTCTGTTTTAGATATCACGTGCTTGATAATGAAACTAACTAGTGATACTACGTAGTTAACTATGAATAGGCCTACTGTAATTCAATAGTGCGTGATATTGAACTAGATTCTGCAACTGCTAATATGCCGTGCTGCACGTTTGGTGATAGTTTAGCATGCTTCACTATAATAAATATGGTAGTTGTAACTACTGCGAATAGGGGGAGTTAATAAATATGATCACTGTGCTACGCTATATGCCGTTGAATATAGGCTATATGATCATAACATATATAGCTATAAGTGATAAGTTCCTGAATATAGGCTATATGATCATAACATATACAACTGTACTATGAATAAGTTAACGAGGA
Figure BDA00002358547601301
(SEQ?ID?NO：#)
Watermark-4338 a uncoded word symbol, 1222 base pairs
CYNTHIA?ANDREWS-PFANNKOCH，QUANG?PHAN，LI?MA，HAMILTON?SMITH，ADI?RAMON，CHRISTIAN?TAGWERKER，J?CRAIG?VENTER，EULA?WILTURNER，LEI?YOUNG，SHIBU?YOOSEPH，PRABHA?IYER，TIM?STOCKWELL，DIANA?RADUNE，BRIDGET?SZCZYPINSKI，SCOTT?DURKIN，NADIA?FEDOROVA，JAVIER?QUINONES，HANNA?TEKLEAB。
"WHAT?I?CANNOT?BUILD，I?CANNOT?UNDERSTAND."-RICHARD?FEYNMAN TTAACTAGCTAATTTCATTGCTGATCACTGTAGATATAGTGCATTCTATAAGTCGCTCCCACAGGCTAGTGCTGCGCACGTTTTTCAGTGATATTATCCTAGTGCTACATAACATCATAGTGCGTGAAAACCTGATACAATAGGTGATATCATAGCAACTGAACTGACGTTGCATAGCTCAACTGTGATCAGTGATATAGATTCTGATACTATAGCAACGTTGCGTGATATTTTCACTACTGGCTTGACTGTAGTCATATGATAGTACGTCTAACTAGCATAACTAGTGATAGTTATATTTCTATAGCTGTACATATTGTAATGCTGATAACTAGTGATATAATCCAACTAGATAGTCCTGAACTGATCCCTATGCTAACTAGGATAAACTAACTGATACATCGTTCCTGCTACGTGATAGCTTCACTGAGTTCCATACATCGTCGTGCTTAAACATCAGTGATAACACTATAGAGTTCATAGATACTGCATTAACTAGTGATATGACTGCAATAGCTTGACGTTTTGCAGTCTAAAACAACGTGATAATTCTGTAGTGCTAGATACTATAGATTTCCTGCTAAGTGATAAGTCTACTGATTTACTAATGAATAGCTTGGTTTTGGCATACACTGTGCGTGCACTGGTGATAGCTTTTCGTTGATGAATAATTTCCCTAGCACTGTGCGTGATATGCTAGATTCTGTAGATAGGCTAAATTCGTCTACGTTTGTAGGTGATAGTTTAGTTGCTGTAACTAATATTATCCTGTGCCGTTGCTAAGCTGTGATATCATAGTGCTGCTAGATATGATAAGCAAACTAATAGAGTCGAGGGGGAGTCTCATAGTGAATACTGATATTTTAGTGCTGCCGTTGAATAAGTTCCCTGAACATGTGATACTGATATTTTAGTGCTGCCGTTGAATATCCTGCATTTAACTAGCTTGATAGTGCATTCGAGGAATACCCATACTACTGTTTTCATAGCTAATTATAGGCTAACATTGCCAATAGTGC (SEQ?ID?NO：#)
Watermark 1-4 replaces respectively box 282-287,447,106 and 680.Watermark is inserted into experiment and shows (watermark 1 (1246bp) and 2 (1081bp)) or predict that (watermark 3 (1109bp) and 4 (1222bp) are the zone of interference cell viability not.All 6 reading frame terminator codons are in beginning and the end of each watermark UnderlineAfc I restriction site shows to add bold Italic.Because the genome sequence that our data indication box 936-939 means is dispensable, so we produce the version of box 940, it comprises with the 80bp of box 935 overlapping.This will produce the 4-kb disappearance and further distinguish synthetic gene group and natural gene group.
Length with synthetic gene group design of this disappearance and 4 watermark sequences is 1,077,947bp.This sequence is separated into has the overlapping length of 80bp 1, the box of 080bp, and NotI restriction site (GCGGCCGC) is added into each end.The identified gene collection of illustrative plates, assemble its 1,078 box, the polymorphism of expectation, unexpected polymorphism, the intestinal bacteria transposon of insertion and other features of mycoplasma mycoides JCVI-syn1, this mycoplasma mycoides JCVI-syn1 of formation provides the Genome Atlas of mycoplasma mycoides JCVI-syn1: gene, structure RNA, watermark, with respect to the polymorphism of natural mycoplasma mycoides capri GM12 and the coordinate of synthetic DNA box.Table 18 is listed the difference between synthetic gene group and contrast natural gene group YCpMmyc1.1 that we clone in yeast.
Table 18
Difference between mycoplasma mycoides JCVI-syn1 and natural gene group YCpMmyc1.1.
Figure BDA00002358547601311
Difference is divided into to two groups: 1) " difference in design "---25 differences between the design of synthetic gene group and natural YCpMmyc1.1 genome, and 2) " off-design "---one that is derived from sMmYCp235 is shifted clone's sequenced genes group and 9 differences of observing between YCpMmyc1.1.Difference classify by type (row 1).Pointed out the coordinate (row 2) of each difference on the mycoplasma mycoides JCVI-syn1 sequence of design.About snps and homopolymer and dinucleotides test, listed the actual sequence difference on synthetic gene group and natural gene group; For watermark, provide the title of watermark and the length of the mycoplasma mycoides sequence pointing out to replace; Size has the deletion and insertion (row 3 and 4) provided.Also show the box of differentia influence and the search word string (row 5 and 6) of each difference of location.
The 1kb box of all designs is by Blue Heron; Bothell, the Washington customization is synthetic.All boxes are separately synthetic and by manufacturers's authentication sequence.
Embodiment 6B
Assembling and the transfer of synthetic donor gene group
Design graduation strategy is to assemble genomes (being illustrated in Figure 20) by conversion and homologous recombination in yeast with 3 stages.In the first stage, once get 10 boxes, to produce the assembling intermediate of 10kb.In subordinate phase, once get 10 these 10kb intermediates, to produce 11 ~ 100kb assembling intermediate.In the end the stage, all 11 DNA fragmentations are assembled into to complete synthetic gene group.
I. The preparation of host's carrier
Use polymerase chain reaction (PCR) amplification, produce for cloning unique carrier of each assembly.The end that the carrier of amplification comprises the assembly end is overlapping.Strategy (Gibson etc., Science 319,1215 (2008)) prepared by the assembling carrier has been described before.That each PCR primer comprises is overlapping with the 20bp of carrier one end, NotI restriction site and 40bp and box-packed part one end overlapping.For the first stage of assembling, produce the yeast/shuttle vehicle that is called the pCC1BAC-LC yeast, for the template DNA of PCR.By external assembling (Gibson etc., Nat.Method., 6:343 (2009)) the PCR product that the pCC 1BAC of AfeI-digestion and, Histidine selective marker overlapping by the 40bp of AfeI restricted fragment, kinetochore and replication origin form builds this carrier and (is derived from pRS313 (Sikorski and Hieter, Genetics 122,19 (1989))).
For the subordinate phase of assembling, pRS314 (Sikorski and Hieter, Genetics 122,19 (1989)) is as the template DNA (difference is the assembling of 831-840, and it uses pCC1BAC as template DNA) of PCR.According to the explanation of manufacturers, by PCR, use
Figure BDA00002358547601331
Hot Start High-Fidelity archaeal dna polymerase and HF damping fluid (NEB) produce the unique fit carrier, and difference is that reaction is supplemented with the other MgCl2 of 1mM and product is annealed under 60 ℃ and every kilobase extends 1min.Extract the PCR product from sepharose after electrophoresis, and, according to the explanation of manufacturers, use QIAquick gel extraction kit (Qiagen) purifying.Although we have used identical subordinate phase carrier for cloning natural and synthetic fragment, build other carrier sequence for cloning natural 100-kb fragment.This carrier sequence called after pCC1BAC-URA and building in the mode identical with building pCC1BACLCYEAST, difference is to use pRS316 to replace pRS313.Primer for generation of PCR-amplification assembling carrier is listed in table 19-21.
Table 19. carries the primer of carrier for generation of unique first stage assembling.
Show overlapping with box sequence end with capitalization.
Figure BDA00002358547601332
Figure BDA00002358547601341
Figure BDA00002358547601351
Figure BDA00002358547601361
Figure BDA00002358547601371
Figure BDA00002358547601381
Figure BDA00002358547601391
Table 20. is for generation of the primer of the synthetic subordinate phase assembling carrier of uniqueness.Show with 10-kb and assemble the overlapping of sequence end with capitalization.
Figure BDA00002358547601392
Figure BDA00002358547601401
Table 21. is for generation of the primer of the carrier of the uniqueness for cloning the natural mycoplasma mycoides fragment of 100-kb.Show overlapping with 100kb native sequences end with capitalization.
Figure BDA00002358547601402
Ii. The assembling of 10kb synthetic mesophase thing
The fragment of donor gene group provides with about equal concentrations as the DNA box, and is included in the escherichia coli cloning carrier.With 10 groups of these boxes of concentrating equivalent, with NotI, digest to discharge inset, gel-purified and then mix with host's carrier---unique yeast/shuttle vehicle---.
A. prepare box DNA
Concentrate the uncut box DNA of about 500ng with 10 groups, with NotI digestion, and under 96V, on 1% low melting-point agarose gel electrophoresis 90min.From gel cutting 1kb DNA fragmentation, and the quality of measurement gel strip.Add the 1:10 solution of the 10 * TAE damping fluid that comprises the 3M sodium acetate to each gel strip, and sepharose melts 10min under 65 ℃.After the gel strip fusing, they are at 42 ℃ of lower incubation 15min, more than adding β-gelase (NEB) and continue incubation 1h with 1:50.Phenol extraction and ethanol precipitation (under 1 μ l glycoblue exists) afterwards, are resuspended in DNA in 40 μ l TE (pH 8.0).10 microlitres are for transformation experiment.
B. host's carrier
This host's carrier is bacterial artificial chromosome (BAC; PCC1BAC[Epicentre]), it has histidine auxotroph mark, kinetochore and the replication origin of insertion, for selecting and propagation at yeast.The Yeast proliferation element has been designed into assembly 831-840.Due to this reason, for the cloning vector of this assembling, only comprise BAC element (pCC1BAC).Increase and utilize the primer comprise with the 40bp overlap of the box end be assembled by PCR-, produce unique first stage assembling carrier (Gibson etc., Science 319,1215 (on February 29th, 2008)).
C. yeast conversion
Also comprise the NotI restriction site in these primers, it makes the assembling box intactly discharge from carrier.Host's carrier/box mixture then is transformed into yeast and incubation a couple of days on the selectivity plate.
In yeast spheroplast conversion process, process cell with zymolyase, to remove cell walls and then to pass through with PEG and CaCl 2Process the ability that produces the picked-up foreign DNA.Before using, disclosed scheme is carried out this process with VL6-48N yeast strain (6), and one of them is improved to: Growth of Cells to OD600 be 0.5 (~ 10 7Individual cell/ml) then prepare the yeast spheroplast.We have found that this optical density(OD) is best for the multiple overlapping fragments of assembling in yeast.Concentrate the NotI digestion fragment, mix (except the terminal stage of assembling) with the unique PCR-amplification of 40ng assembling carrier, and then be added into ~ 2 * 10 8The yeast spheroplast.After transforming, the yeast spheroplast is not having Histidine (CSM-His; The 10kb assembly, 811-900, and complete genome group) or there is no tryptophane (CSM-Trp; The 100kb assembly, except 811-900) the full complement substratum and 1M Sorbitol Powder agar plate under 30 ℃ regeneration and select 3 days.The initial conversion body is then transferred on the selectivity plate and under 30 ℃ and is incubated overnight as small pieces.
D. the screening of subordinate phase assembly and preparation
Intestinal bacteria are extracted and be converted into to plasmid DNA from single yeast clone, and it is the more stable host of assembling box propagation.The cell (gels data shows successful 10kb assembly: data do not show) of the carrier that comprises the 10kb inset with assembling is then separated and digest to screen with NotI to plasmid DNA from single escherichia coli cloning.
According to the explanation of manufacturers, use QIAprep Spin Miniprep test kit (Qiagen) from ~ 10 7Yeast cell (from spot) extracts DNA, one of them improvement is that damping fluid P1 is supplemented with 1:1000 beta-mercaptoethanol and 1:10020mg/ml Zymolyase-100T (USB), and cell, at 37 ℃ of lower incubation 30-60min, then adds damping fluid P2.With 1,200V, 25 μ F and 200 Ω, use Gene Pulser Xcell Electroporation system (BioRad), the sample of yeast-extraction DNA (3 μ l at the most) is converted into 30 μ lEPI300 in 1-mm Glass tubing (BioRad) TM(Epicentre) electricity turns competence (electrocompetent) Bacillus coli cells.Make cell recover 1.5h in 37 ℃ of lower 1ml SOC substratum, then bed board is on the LB substratum that comprises 12.5 μ g/ml paraxin.After 37 ℃ of lower incubation 16-24h, select single clone and under 37 ℃, there is grow overnight in the 3mlLB substratum of 12.5 μ g/ml paraxin.
From these cells, by using the alkaline bleach liquor cleavage of P1, P2 and P3 damping fluid (Qiagen), then isopropanol precipitating, prepare DNA.The DNA bead is dissolved in the TE damping fluid (pH 8.0) that comprises RNase A and RNase T1 (Ambion).Alternatively, according to the explanation provided, from QIAprep Spin Miniprep test kit (Qiagen), prepare DNA.
After purifying, with NotI (with using once in a while SbfI) DNA digestion, from carrier, to discharge inset, also by gel electrophoresis 30-60min 0.8%E-gel (Invitrogen), arrange by size.Use Amersham Typhoon 9410 fluoroscopic image instrument to make the band video picture.
Positive colony is bred and is incubated overnight under 37 ℃ in the 10ml LB substratum that comprises 12.5 μ g/ml paraxin and 1:1000 inducing solution (Epicentre).Collect culture, and use the explanation purify DNA molecule of QIAprep Spin Miniprep test kit (Qiagen) according to manufacturers.Assembly 211-220 is at Epi300 TMIn bacterial strain, be unsettled, so it is transferred to Stbl4 bacterial strain (Invitrogen), it can stably maintain there.This assembly can not be induced to higher copy level in this bacterial strain, and can not be as above by post-purifying.In fact, this is cloned in the 50ml LB substratum that comprises 12.5 μ g/ml paraxin and breeds.In and after the cell of cracking, centrifugal these DNA moleculars, then use isopropanol precipitating.The DNA bead is dissolved in to then RNase processing in TE damping fluid (pH 8.0), and phenol chloroform (phenolchloroform) extracts and the ethanol precipitation.The DNA bead is dissolved in the TE damping fluid.For each 10kb assembly, the DNA of NotI-digestion is by quantizing with the gel electrophoresis side by side of known dna standard substance.
Generally speaking, can obtain the fragment of at least 1 10kb assembling by screening 10 yeast clones.But success ratio changes between 10-100%.1 assembly, 791-799, can not produce by homologous recombination in yeast.This fragment is divided into two parts, 791-795 and 796-799, and it assembles separately (Gibson etc., NatMethods 6,343 (in May, 2009)) by vitro recombination.Except 211-220, all first stage assemblies are bred in Epi300 Bacillus coli cells (Epicentre).This clone allows cloning vector is induced to high copy number from single copy.Assembly 211-220 is unstable in the Epi300 cell, so be transferred to the Stbl4 cell, it can stably maintain there.
To all first stage intermediate order-checkings.19 in 111 assemblies comprise mistake.Each comprises the single error that comes from respectively box 82 and 812 our sequential analysis announcement assembly 81-90 and 811-820.Box 82 is corrected and is ressembled to produce faultless clone 81-90 in the 1-kb level.It is 811 overlapping that sudden change in box 812 occurs in, and it does not comprise mistake.So, when the clone of the 811-820 to other is sequenced, some in these do not comprise mistake.The 811-820 clone of 1 sequence-checking is used for assembly reaction subsequently.We select not proofread and correct the mistake existed in 121-130, because it is the same sense mutation in dispensable gene.This sudden change is used as other variation further to distinguish synthetic gene group and natural gene group (table 18).Can avoid a sudden change by maintain the assembling fragment between proliferation period in intestinal bacteria at single copy number.By connecting at a box, (intersection junction) not exclusively removes the NotI restriction site and can produce a mistake.Four mistakes may be derived from the primer for the pcr amplification cloning vector.Remaining mistake produces between proliferation period in yeast.Select and check order and confirm the optional clone of 15 assemblies.
Iii. The assembling of 100kb synthetic mesophase thing
A. prepare DNA
With 10 groups of 10kb assembling intermediates of concentrating equivalent, digest to discharge inset with NotI, and follow gel-purified.The uncut DNA of about 125ng concentrates with 10 groups, with NotI digestion (usually also with SbfI, digesting to provide the better separation between carrier ribbon and inset) and under 96V on 1% low melting-point agarose gel electrophoresis 90min.From 10 kb DNA fragmentations of this gel cutting, and extract after processing as above-mentioned β-gelase.DNA is resuspended in 20 μ l TE (pH 8.0).10 microlitres are for being used the transformation experiment of 40ng carrier.
B. host's carrier
The assembly of these gel-purified mixes with unique subordinate phase host's carrier, and it as above prepares, except pRS414 (Sikorski and Hieter, Genetics 122,19 (in May, 1989)) is used as pcr template.Because in yeast, selectable mark will be changed into tryptophane from Histidine, we infer that the background of not expecting the clone will be lowered.The Yeast proliferation element will be Already in assembly 811-900.So 831-840 is the same with assembly, the cloning vector of assembly 811-900 only is comprised of the BAC element.
C. yeast conversion
Concentrated 10-kb assembly and their cloning vectors separately as above are transformed into yeast, to produce 100kb assembling intermediate.This produces hundreds of yeast clones.
D. screen and prepare final donor gene and assemble part
For showing the expectation PCR pattern of each 100-kb assembly, the multiple PCR primer of listing in table 22 is for screening YCpMmyc1.1, and it extracts from yeast.The spaced apart 100bp of pcr amplification.
Table 22
Figure BDA00002358547601441
Except amplicon, 10d is (underlined; This zone is corresponding to 931-940) and assembly 796-799, all first stage assemblies can pass through this pcr analysis explanation.Downstream analysis is for further confirming 701-799 and 901-100 intermediate.Result indication mycoplasma mycoides 100kb assembling intermediate can not stably remain in intestinal bacteria, so may not adopt for screening the method for 10kb assembly.In fact, it is 110 primer pairs from the amplicon of 100bp to 1150bp that design produces magnitude range, so that they can produce 9-11 amplicon in each of 11 independent multi-PRC reactions; Each 100kb assembling intermediate is one (table 23 and table 24).
Table 23: amplicon
Figure BDA00002358547601451
Table 24: for the identification of the multiple PCR primer of the 100-kb intermediate that comprises all first stage assemblies.
Figure BDA00002358547601452
Figure BDA00002358547601461
Figure BDA00002358547601471
Figure BDA00002358547601481
Figure BDA00002358547601491
Because each 10kb assembling intermediate is meaned by primer pair, so the existence of all amplicons can be pointed out the 100-kb intermediate of assembling.DNA extracts and passes through the multiplex PCR assay with primer pair separately from 10 or more yeast clone.As above from sheet (patch), extract DNA.
Use the Qiagen multiple PCR reagent kit, the primer that in use, table 24 is identified carries out multiplex PCR.10 * primer raw material of 20 kinds of oligomers that the DNA extraction thing that comprises 1/50 volume (1 μ l) in each 10 μ l reaction and 1 μ l comprise every kind of 2.5 – 5.0 μ M.Loop parameter is 94 ℃, 15min, and then 35 circulations: 94 ℃, 30s; 57-60 ℃, 90s; With 72 ℃, 90s then carries out 3 minutes incubations of single under 72 ℃.Then, each reaction application of sample to 2% of 2 μ l
Figure BDA00002358547601502
(Invitrogen) upper, and apply 72V 30min.Band is used Typhoon 9410 fluoroscopic image instrument video pictures.
For separate on a small scale 100kb superhelix ring-type assembly from yeast, the yeast culture that 5ml is saturated is grown in selective medium.The method (Leem etc. (2008) Genome, 51:155) of this process based on describing before, but scale reduces and do not emphasize to remove yeast chromosomal dna.
Briefly, the yeast cell of results is transferred to the Eppendorf tube with 1ml water, and is resuspended in the 1ml pre-treatment damping fluid (1.2M Sorbitol Powder, 200mM Tris-Cl, 100mM EDTA, pH to 9.1) with 8 μ l 14M beta-mercaptoethanols.At room temperature after incubation 10min, harvested cell, with 1ml SCE (1M Sorbitol Powder, 60mM EDTA, the 100mM Trisodium Citrate, pH 5.75) rinsing twice, and be resuspended in 1ml SCE add 10 μ l Zymolyase-100T solution (20mg/ml Zymolyase-100T[USB], 50% glycerine, 2.5% glucose, 50mM Tris-Cl, pH 8.0) in.At 37 ℃ of lower incubations after 1 hour, the results spheroplast, and then be resuspended in 25 μ l Tris/ sucrose damping fluids (50mM Tris-Cl pH 8.0,25% sucrose) and 20 μ l Proteinase K solution (10mg/ml Proteinase K [Sigma], 1mM calcium chloride, 50mM Tris-Cl pH 8.0).Next, add 475 μ l lysis buffers (1% SDS, pH 12.45 for 20mM EDTA, 50mM Tris-Cl) and by upper and lower imbibition mixing.After 37 ℃ of lower incubation 30min, add 100 μ l 2M Tris-Cl pH 7.0 and mix.Next, add 100 μ l 4M NaCl and mix.At room temperature, after incubation 30-min, add 70 μ l 3M sodium acetates and mix.Carry out phenol-chloroform extraction, isopropanol precipitating and the 70% ethanol rinsing of standard and the DNA bead is resuspended in and comprises RNase A, in the 100 μ l TE of pH 8.0.At 37 ℃ of lower incubations, after 1 hour, 10 μ l DNA application of samples are to 1% sepharose in 1X TAE damping fluid and carry out electrophoresis 3 hours with 4.5V/cm.After electrophoresis, use Gold dyeing gel also scans with GE Typhoon 9410 imagers.
Generally speaking, 25% or the clone of more screenings comprise all amplicons for complete assembly expection.Select one of these clones for further screening.Extract cyclic plasmid DNA and with superhelix mark sepharose side by side on arrange by size.The length of subordinate phase assembly with success of carrier sequence is about 105kb (data do not show).While after multiplex PCR, producing all amplicons, usually produce the subordinate phase assembling intermediate with correct size.But, in some cases, little disappearance occurs.In other cases, assemble multiple 10kb fragment, it produces larger subordinate phase assembling intermediate.Fortunately, these differences can, before completing the genome assembling, easily be found on sepharose.
Iv. The final assembling of synthetic donor gene group
A. prepare DNA
In the assembly of preparation terminal stage, separate every kind of 11 kinds of subordinate phase assemblies of Microgram; This is important, because these assemblies must be purified into from yeast.As (Devenish and Newlon, the Gene 18,277 (June nineteen eighty-two)) that report before, we find that the cyclic plasmid of our subordinate phase assembly size can separate after the alkaline bleach liquor cleavage process from the yeast spheroplast.For being further purified 11 kinds of assembling intermediates, they are processed and pass through ion exchange column by exonuclease.Total plasmid DNA (1/100th) of small portion digests with NotI and passes through field inversion gel electrophoresis (FIGE) and analyze (data do not show).The every 400ml yeast culture (~ 10 of the method 11Individual cell) produce every kind of assembly of ~ 1 μ g.
For the extensive 100kb assembly that separates from yeast, will comprise preculture thing (5 to the 10ml) grow overnight of every kind of VL6-48N bacterial strain of one of 11 100kb assemblies with saturated in selective medium and then be seeded in the selective medium of 400ml.Once culture reaches 1.5 OD600, harvested cell (2,205rcf, 3min).Be resuspended in 100ml water by bead and be transferred in 2 50ml conical tubes and as above gather in the crops.Cell pellet is resuspended in the 40ml SPE that comprises 0.4ml Zymolyase-20T (10mg/ml) and 40 μ l 14M beta-mercaptoethanols (the 0.01M sodium phosphate, pH 7.5 for 1M Sorbitol Powder, 10mM Na2EDTA).Cell suspending liquid is at 37 ℃ of lower incubation 60min.Results spheroplast (1,125rcf continues 5min) also is resuspended in 1ml 1M Sorbitol Powder, and then adds the lysis buffer (0.05M Tris-HCl, 0.02M EDTA, 1%SDS, pH 12.8) of 20ml and reverse and manage 10 times.At 37 ℃ of lower incubation cellular lysate 30min, and then at 20 times, reverse gently and, after the centrifugal 20min of 3,645rcf, extract by 20ml phenol/chloroform/isopentyl-ol (Invitrogen).
Water is transferred to new 50ml conical tube.By adding the Virahol of 2ml 3M NaAc (pH 5.2) and 20ml, follow under 4 ℃ with the centrifugal 30min of 3,645rcf, carry out the DNA precipitation.Bead is resuspended in the 1ml TE (pH 8.0) that comprises RNase A (30 μ g/ml) and at 37 ℃ of lower incubation 30min.Now, combine two DNA samples.Be further purified cyclic DNA by Qiagen Large-Construct test kit.According to the explanation of manufacturers, use some less improvement to carry out purifying.The EX damping fluid of 10ml mixes with DNA solution, then adds 200 μ l Exonuclease and 300 μ l 100mM ATP.After 37 ℃ of lower incubation 45-min, by adding the QS damping fluid termination reaction of 12ml.This solution then is applied to Qiagen-tip 500 posts.With this post of QF damping fluid rinsing of 30ml, and with 12ml QF buffer solution elution DNA, 55 ℃ of lower preheatings.Under the existence of the GlycoBlue (Ambion) followed at 1.2ml 3M NaAc (pH 5.2), 15 μ l and the total tRNA of yeast (Sigma) of 15 μ l, by adding the ethanol of 2 volumes, under-20 ℃, precipitation DNA spends the night.
Alternatively, by adding 1 volume isopropanol precipitating DNA.By with 3,645rcf, under 4 ℃ centrifugal 1hr, reclaim the DNA of precipitation.Ethanol rinsing DNA bead with 70% also is resuspended in 150 μ l TE (pH 8.0).Each of sample (0.75 μ l) digests and analyzes by U-2FIGE with NotI.Not under loop condition at 1% sepharose (BioRad of 1 * TAE damping fluid, article No. 161-3016) carry out the FIGE analysis on, and parameter is forward 90V, initial conversion 5.0sec, finally change 30sec, linear slow the liter, with reverse 60V, initial conversion 5.0sec, finally change 30sec, linear slow the liter.After electrophoresis, use
Figure BDA00002358547601521
Gold dyeing gel also scans with GE Typhoon9410 imager.
B. yeast conversion
Top method is not removed all linear yeast chromosomal dnas fully, and we find that it can significantly reduce yeast conversion and efficiency of assembling.For further abundant 11 kinds of ring-types assembling intermediates, concentrate every kind of assembly ~ 200ng sample mixing with the agarose of fusing.Along with agarose solidifies, fiber is through also topological " catching " cyclic DNA (Dean etc., Anal Biochem 56,417 (in December, 1973)).The linear DNA of not catching can be followed electrophoresis and leave the agarose inserted block, so the enrichment ring molecule of catching.11 kinds of ring-type assembling intermediates digest with NotI, thereby can discharge inset.Subsequently, from the agarose inserted block, extract fragment, analyze (data do not show) by FIGE, and be transformed into the yeast spheroplast.The 3rd and final stage in assembling, do not need other carrier sequence, and this is because Yeast proliferation element Already in assembly 811-900.After incubation, approximately 100 colonies appear on the selectivity flat board.
Topology is caught and is analyzed
Concentrate every kind of uncut 100kb assembly the balance to 50 ℃ of 20 microlitres.2% low melting-point agarose of 1 volume (220 μ l)---also balance to 50 ℃, mix with concentrated DNA.About this mixture of 85 μ l is added into to the agarose inserted block mould (Bio-Rad) that keeps ice-cold on ice.After incubation 30min on ice, the agarose inserted block be added in the hole of 1% sepharose to (1 * TAE damping fluid) and carry out electrophoresis 2 hours with 4.5V/cm.Inserted block shifts out and passes through and at room temperature in 5ml 0.1 * rinsing damping fluid (Bio-Rad CHEF genomic dna inserted block test kit), reverses and within 1 hour, carry out rinsing from sepharose.Remove this damping fluid and add 5ml 1 * damping fluid 3 (NEB).At room temperature incubation/after reversing 1 hour, the removal damping fluid also adds the fresh 1 * damping fluid 3 (2.5ml) with 250 NotI of unit.By at 37 ℃ of lower incubation agarose inserted blocks, spending the night and carry out NotI digestion.
In 5ml 1 * TAE/0.3M sodium acetate solution, reversing agarose inserted block is 1 hour.The agarose inserted block then moves to Eppendorf tube.10 * TAE damping fluid 1:10 (~ 40 μ l) that will comprise the 3M sodium acetate is added into gel strip, and after 50 ℃ of lower initial incubation 15min, fusing sepharose 7min under 68 ℃.After gel strip fusing, they are 42 ℃ of lower incubations 15 minutes, more than 1:50 (~ 8 μ l) adds β-gelase (New England Biolabs) and continues incubation 1h.After the ethanol of phenol extraction gently (by reversing pipe 10min lentamente) and standard precipitates (under the existence of 1 μ l glycoblue), DNA is resuspended in 20 μ l TE (pH 8.0).Almost 0.5 μ l is for transformed yeast.This 0.5 μ l sample is as above analyzed by U-2FIGE.
Yeast agarose inserted block
Yeast culture (50ml) adds in VITAMIN B4 substratum (Teknova) and grows to 1.0 OD600 at CSM-His.The results culture is also then used the 50ml water rinse.Next, the results culture is also then with 10mlEDTA pH 8.0 rinsings.Gather in the crops this culture and then be transferred in the Eppendorf tube with 750 μ l cell suspension damping fluids.Cell pellet then is resuspended in to 150 μ l cell suspension damping fluids.By this mixture balance to 50 ℃ and then with 85 μ l Zymolyase-100T solution (20mg/ml Zymolyase-100T[USB], 50% glycerine, 2.5% glucose, 50mM Tris-Cl, pH 8.0) and 225 μ l 2% low melting-point agaroses mixing.About this mixture of 85 μ l is added into to the agarose inserted block mould (Bio-Rad) that keeps ice-cold on ice.After incubation 30min on ice, inserted block is added into to the Lyticase damping fluid (10mM Tris-Cl pH 7.5,50mM EDTA pH 8.0) that 5ml comprises 500 μ l Zymolyase-100T solution.After 2 hours, use 25ml water rinse inserted block at 37 ℃ of lower incubations.Bio-Rad CHEF genomic dna inserted block test kit is used for carrying out Proteinase K incubation and rinse step, and is described in the handbook provided with test kit.
C. screen and analyze synthetic donor gene group
In order to screen the complete genome group, carry out multiplex PCR with 11 pairs of primer pairs, it produces the amplicon (table 25) of 11 kinds of magnitude range from 337bp to 1149bp in a reaction.
Table 25. is for the identification of the genomic multiple PCR primer of complete assembling.This primer sets is called TSS2; Each primer sets one of connects across 11 kinds of 100-kb.
Figure BDA00002358547601531
Figure BDA00002358547601541
The multiplex PCR that produces 11 kinds of amplicons by use screens the yeast clone of the synthetic gene group that comprises complete assembling; The everywhere connected at 11 kinds of assemblies is a kind of.The design primer pair is to cross over each that 11 kinds of 100-kb assemblies connect.Just 48 clones of screening, extract DNA from a clone (sMmYCp235) who produces all 11 kinds of amplicons.The PCR of WT positive control (YCpMmyc1.1) produces undistinguishable 11 kinds of amplification subgroups (data do not show).For further showing the synthetic genomic complete assembling of mycoplasma mycoides, in the agarose inserted block from the yeast separation global DNA and carry out two restriction analysis; AscI and BssHII.Because these restriction sites are present in 3 of 4 watermark sequences, so this digestion selects to produce the unrestricted model (Figure 20 and table 26) different from natural mycoplasma mycoides genome.Natural (WT) and synthetic (235) mycoplasma mycoides genome in the agarose inserted block from yeast separation.In addition, separately from the host strain purify DNA.The agarose inserted block is with AscI or BssHII digestion and by clamper uniform electric field (CHEF) gel electrophoresis isolated fragment.
The genomic restriction analysis of the mycoplasma mycoides of breeding in yeast
Twice 1hr of rinsing yeast agarose inserted block be supplemented with balance 1hr in 1ml1 * damping fluid 2 of BSA (NEB) in 0.1 * rinsing damping fluid (Bio-Rad) of 1ml.Yeast chromosomal dna is with every kind of restriction enzyme AsiSI of 50 units and RsrII digestion spend the night (these enzyme indigestion mycoplasma mycoides genomes).Inserted block is followed application of sample on the 1%TAE sepharose so that the pastoris genomic dna fragment electrophoresis of digestion goes out inserted block (ring-type mycoplasma mycoides genome is still in inserted block).Sepharose was with 6V/cm electrophoresis 3 hours.After electrophoresis, the agarose inserted block shifts out and twice 1hr of rinsing at 1 * damping fluid, the 3 (NEB of 1ml in 0.1 * rinsing damping fluid of 1ml from hole; For BssHII digestion) or 1 * damping fluid, 4 (NEB; For AscI digestion) balance 1hr.The mycoplasma mycoides genomic dna spends the night with the restriction enzyme BssHII of 50 units or the AscI digestion of 50 units.After incubation, at room temperature rinsing of 1ml 0.1 * rinsing damping fluid 1 hour the application of sample horizontal pulse field gel electrophoresis of going forward side by side on sepharose for all inserted blocks.All restriction enzymes are bought from NEB.
Pulsed field gel electrophoresis
Yeast agarose inserted block (Fig. 4 c) and mycoplasma mycoides agarose inserted block (Fig. 5 b) utilize clamper uniform electric field (CHEF DR III; Bio-Rad) circulate under 14 ℃ and experience pulsed field gel electrophoresis in 1% sepharose of 1 * TAE with 0.5 μ g/ml ethidium bromide.Under 4.0V/cm, pulse number delays and rises to 120s from 60 at 20-24hr.
Separation is from the genomic sequential analysis of the mycoplasma mycoides JCVI-syn1 of mycoplasma mycoides JCVI-syn1 cell.
Cell is grown in the SP4 substratum that comprises the 10mg/l tsiklomitsin, and uses the Promega genome DNA extracting reagent kit, extracts genomic dna.The combination of use 454 and Sanger technology, sequenced dna.Use order-checking pcr amplification and the direct gene group that cover breach to walk the closed breach of the combination of looking into.Use CeleraAssembler assembling genome.The sMmYCp235 clone produces the unrestricted model (data do not show) of complete assembling synthetic gene group expection.
Table 26
Figure BDA00002358547601542
Figure BDA00002358547601551
Natural (WT) and the Asc I of synthetic (Syn235) mycoplasma mycoides expectation and the size of BssH II restricted fragment have been shown.
V. Shift synthetic donor gene group and enter recipient cell
The other agarose inserted block used in gel analysis in the above also is used in the genome shift experiment.Complete synthetic mycoplasma mycoides genome from the sMmYCp235 yeast clone is transferred to the negative mycoplasma capri recipient cell of restricted type, (Lartigue etc., Science 325,1693 (on September 25th, 2009)) as described earlier.By under 37 ℃, be chosen in the blue colony of growth on the SP4 substratum that comprises tsiklomitsin and X-gal, to result, score.Separation produces the blue colony of 5-15 tetracycline resistance from each agarose inserted block of genome of this yeast clone.This can be comparable to the YCpMmyc1.1 contrast.When the two exists when mycoplasma capri recipient cell and mycoplasma mycoides genome, observe the recovery of colony in all shift experiments.
In order to distinguish fast synthetic body and mycoplasma capri or the natural mycoplasma mycoides of shifting, carry out two analyses.At first, design each specific 4 primer pair to 4 watermarks, thus they produce 4 kinds of amplicons (table 27) in single multi-PRC reaction.
Table 27
Figure BDA00002358547601552
Produce the multiplex PCR of the primer sets of 4 kinds of amplicons by use, the transfer body that screening comprises the synthetic gene group; One is each inside of 4 watermarks.Analyze side by side with natural, the non-synthetic gene group (WT) that migrates out yeast a transfer body (syn1) that comes from yeast clone sMmYCp235.
Produce all 4 kinds of amplicons (data do not show) by the transfer body of---rather than YCpMmyc1.1---generation from sMmYCp235.
The second, used the gel analysis of AscI and BssHII as above-mentioned.In brief, natural (WT) shifts body with the mycoplasma mycoides of synthetic (syn1) mycoplasma mycoides genome from the agarose inserted block and separates.With AscI or BssHII digesting agar sugar inserted block and separate fragment by the CHEF gel electrophoresis.By the corresponding correct big or small restricted fragment of the fragment number indication shown in table 25.The unrestricted model obtained and the transfer body consistent (data do not show) produced from synthetic mycoplasma mycoides genome.
Order-checking is derived from the single transfer body of sMmYCp235 synthetic gene group.Except the known polymorphism occurring during building-up process and 8 new polymorphisms and the intestinal bacteria transposon do not expected insert, the design of sequence and expectation is mated.This bacterial strain is called mycoplasma mycoides JCVI-syn1.Colony is grown on the SP4 agar that comprises Xgal, so that express the cell of β-cow's milk Glycosylase, becomes blue.The two diameter of JCVIsyn1 and wild-type colony is 0.5mm.In the haematolysis ability test of JCVI-syn1 and wild-type biology, colony is with comprising the erythrocytic Agar overlay of 5% Sheep Blood.The cracking of wild-type mycoplasma mycoides is the cell below colony directly.The feature that the agar of this pattern and micro-green is α haemolysis.Not cracking of mycoplasma mycoides JCVI-syn1 colony erythrocyte.
Shift and do not have different from wild-type mycoplasma mycoides subspecies capri on surface.Colony growth and morphology speed does not have difference (data do not show); But we plan that (program) makes the synthetic gene group from different on wild-type mycoplasma mycoides capri phenotype really.This mycoplasma kind is goat but is not people's opportunistic pathogen (DaMassa etc., Am J Vet Res 44,322 (February nineteen eighty-three)).Think that toxic mechanism is that bacterium produces hydrogen peroxide, the tissue of its infringement infection animal.In mycoplasma mycoides, hydrogen peroxide is the by product of glycerol metabolism.For reducing the pathogenic potential of our synthetic gene group, we omit gene (gtsA, gtsB, gtsC, and gtsD) (P.Pilo etc., the J Bacteriol 187,6824 (in October, 2005) of coding mycoplasma mycoides ABC glycerine transporter; E.M.Vilei, J.Frey, Clin Diagn Lab Immunol 8,85 (January calendar year 2001)).As this elliptical result, not cracking of mycoplasma mycoides JCVI-syn1 Sheep Blood red corpuscle, and wild-type biology shows the α Hemolysis characteristic (data do not show) of erythrocyte hydrogen peroxide infringement.Haemolysis is the standard test of mycoplasma toxicity.
Embodiment 6C
Semi-synthetic donor gene assembling is joined and shifts
Because any mistake in genome may not produce the transfer body, so development approach is to verify independently the viability of the synthetic assembly of single 100-kb.For helping the function of the synthetic fragment of each 100-kb of test, build and shift semi-synthetic genome.By natural fragment is mixed with synthetic fragment, can verify the successful structure of each synthetic 100-kb assembly, and these intermediates that needn't check order.By the method (Leem etc., Nucleic Acids Res 31, e29 (on March 15th, 2003)) of describing before using, we have cloned 11 kinds of overlapping natural 100-kb assemblies in yeast.In 11 parallel reactors, with the mycoplasma mycoides genomic dna (YCpMmyc 1.1) of the fragmentation of length average out to ~ 100kb be designed to the PCR-amplification vector cotransformation yeast cell of the end of overlapping 100kb inset.For keeping suitable overlapping, can be reorganized with synthetic fragment so that natural, use the overlapping primer of identical 40-bp had for cloning the synthetic assembly of 100-kb to produce the carrier of PCR-amplification.The 2-10 kind that the semi-synthetic genome built comprises 11 kinds of 100-kb synthon assemblies.Once produce the colony that can survive after shifting, determine that each genomic composite part does not comprise lethal mutation.Obviously, not survival of only a kind of---811-900---in the 100-kb sub-assemblies.
At first, the above-mentioned 811-820 clone who comprises mistake is for generation of not being the synthetic gene group that shifts body.This expects, because single base pair disappearance produces the frameshit for chromosome duplication indispensable gene dnaA.Not to be noted this sudden change before us.By using semi-synthetic genome to build strategy, we can identify the source of 811-900 for not synthesizing shift experiment fast.Therefore, we start to ressemble inerrancy 811-900 assembly, and it is for generation of the sMmYCp235 yeast strain.The genome of dnaA-sudden change and the synthetic gene group in sMmYCp235 be Nucleotide of difference only.In our shift experiment, this genome is as negative control.The dnaA sudden change is also repaired (Noskov, Segall-Shapiro and Chuang, Nucleic Acids Res, (on March 12nd, 2010)) by the genome project in yeast in the 811-900 level.The 811-900 assembly of repairing is used in the genomic yeast clone that has reparation in the terminal stage assembling with generation.This yeast clone called after sMmYCP142 also can be transferred.From the genomic complete list of 11 fragment assemblings success transfer, be provided at table 28.
Table 28
The genome that assembling success are shifted from 11 fragments
Embodiment 6D
The checking of box, assembling intermediate and mycoplasma mycoides JCVI-syn1
Because synthetic cell genome structure scheme is followed hierarchical layered (tiered hierarchy) (1-kb box, 10-kb sub-assemblies, 100-kb sub-assemblies and complete synthetic gene group), so carry out the quality control effort, there is the sequence that is designed to have with the DNA that guarantees to be assembled.Because variable costs restriction, the time limitation changed and the intrinsic difference in order-checking---have many zonules of DNA of overlap from the zone of a large nonrepetitive DNA of order-checking, each level is used different sequencing strategies.Fundamentally different on each different levels the easiest, the most most economical and that the sequence verification method is made great efforts at structure the most fast.And the variable restriction of these methods causes all modes of suddenling change to continue the 100-kb level in the minority of box level introducing.And the assembling of each level is relevant to some sudden changes of generation under a few cases.Need the quality control effort under these all levels that show to make great efforts at the synthesising biological of attempting to build large DNA zone.
The checking of 1-kb box.
Box is bought from Blue Heron.Blue Heron also provides the sequential tracks file, and it confirms the sequence of the DNA of conveying; These trace files are processed in semi-automatic mode.Inner base judges that software (in-house base-calling software) is for becoming the fasta form from the trace file abstraction sequence.Use the ClustalW (Larkin etc., Bioinformatics 23:2947 (2007)) of default setting that gained sequential file and target reference sequences are compared.Gained is compared in pairs with the PERL script of writing for this purpose and is analyzed, and the sequence location of target reference sequences is not mated in identification.Because the trace file that each 1-kb box is provided by two or more Blue Heron covers, it produces again as many paired comparison, so the list of the mismatch position of all paired comparisons of concrete 1-kb box is consistent with each other from covering.This realizes by another kind of conventional PERL script, and then when all readings of consideration manual analyzing be different from the box position of target sequence.
In 1031 boxes, 578 integral body are passed through automatic screening.In remaining 453 boxes, 370 have difference, and it is divided into and is judged as the classification that does not need direct manual analyzing trace file.Two such classification are arranged: 1) significantly sequence to deviate from be in the NotI site of box flank.Sanger order-checking performance due to the difference that only connects the sequencing primer back, so this is common, but this is without worry, because the integrity in NotI site can be detected immediately between the normal epoch of handle box, it relates to NotI cloning vector is cut out to box, and gel-purified.2) some BlueHeron trace files comprise wide C peak usually, because the oxidation of-dyestuff terminator reactant large with C-is relevant sometimes.As a result, some trace files produce incorrect C base judgement.In 453 boxes not by 83 (83) individual their trace files of checking by naked eyes of automatic Verification; 62 are considered to not need inside again to check order, and 21 are checked order again.In final analysis, 3 1-kb boxes are measured has sudden change (2 mononucleotides disappearances and 1 mononucleotide replacement), in only finding those between the trace file screening still provided at Blue Heron one.Fail in the 1-kb level to survey other two mistakes, be mainly that the minority due to poor quality reads (have unusual wide C peak those), and in one case, send (pay, deliver) incorrect clone.All 1-kb boxes by this screening are for generation of the 10-kb sub-assemblies.
The checking of 10-kb assembling intermediate.
Concentrate 10-kb sub-assemblies 454 sequence verification through using non-paired end to read.Originally, screen 116 10-kb assemblies.These 116 assemblies comprise some 10-kb sub-assemblies, and it is double or almost double.Because optional clone's parallel testing of some 10-kb sub-assemblies, so be such situation.And the optional version of some identical 10-kb sub-assemblies is by parallel testing except the existence of variation such as watermark of some design.Because need double or the almost existence of double sequence of independently reading, and because overlapping in adjacent 10-kb sub-assemblies, 80 base pairs have the double zone, 116 10-kb sub-assemblies concentrate in 4 storehouses that separate, and it prevents in storehouse that any 10-kb sub-assemblies is identical at one of two 10-kb sub-assemblies with at its two ends, sharing 80 base pairs.The concrete 10-kb fragment of all optional clones and optional version is separated from each other.And, there is each other some 10-kb sub-assemblies of height similar sequence, such as comprising those of IS1296 insertion sequence, in 4 storehouses, be separated from each other as much as possible.4 storehouses barcode that is encoded, then concentrate in the storehouse by 454 order-checkings.Then separately from the reading of the storehouse of 4 barcodes that are encoded, and use the high-throughput of application defaults setting to be analyzed with reference to the CLC Workbench software package of installation kit group.The storehouse separated by the test of 500N for this, artificial constructed " reference sequences " formed by 29 10-kb target sequences with reference to (against), assembling reading from each storehouse.After the assembling reference sequences, use the mixing of manual observation, CLC SNP prospecting tools and CLC DIP prospecting tools to survey sudden change.CLC does not have the automated manner of surveying difference between reference sequences and consensus sequence.In order to walk around this defect, use the following non-default setting to SNP and DIP instrument: minimum covering=1, maximum covering=999999, minimum variation frequency=50% or counting=999999.Every other the default of them that be set to.Originally, find 18 variations that comprise from they target sequences of 116 10-kb sub-assemblies.Finally, the mark in determine that 3 of these variations are to be cloned by the 10-kb sub-assemblies synthetic gene group of---it has the frame sequence that pollutes the 10-kb sequence---and the sequence similarity between the mark in the plasmid skeleton cause.Find that the plasmid that in intestinal bacteria, 10-kb clones after the selection clone's that several remaining variations and directed PCR and order-checking directly separate with intestinal bacteria from inducing from yeast, intestinal bacteria from inducing Sudden change region induces relevant.The 1 class sudden change of observing 3 times is to introduce " GC " seeing when overlapping.Infer that this may be residual in the NotI site of box overlap flank.In manufacturing they attempt first the optional assembly of the 10-kb fragment of sudden change then as before checked order, except carrying out in the storehouse of two encoding strip shape codes only specifically.At the 10-kb level detection to 1 of 3 sudden changes of 1-kb box.Not to be induced the every other sudden change that intestinal bacteria cause to come from structure.
The checking of 100-kb assembling intermediate.
Replace every kind of 100-kb sub-assemblies of order-checking, build semi-synthetic genome.The 2-10 kind that these semi-synthetic genomes comprise 11 kinds of 100-kb sub-assemblies, and remaining genome is derived from natural mycoplasma mycoides genome sequence.These semi-synthetic genomes are successfully shifted, and confirm does not have lethal mutation in genomic composite part.Become apparent very soon, only a kind of 100-kb sub-assemblies can not be survived: its corresponding box 811a-900.Because this 89-kb fragment can not be is easily obtained in a large number from yeast---easily from yeast a large amount of acquisitions will make direct gene group step look into or 454 order-checkings easily or cost effective, so generate overlapping pcr amplification, and every 200 base pairs of the every kind of amplicon replaced along interchain check order with sequencing primer.
Show the genomic respective regions of natural mycoplasma mycoides that can survive the two carries out this process for synthetic 811a-900 with in semi-synthetic experiment.Again the natural fragment that checks order guarantees to avoid occurring this possibility: the 811a-900 zone has been designed will prevent the deviation of viability when shifting.And the synthetic 811a-900 that checks order is for guaranteeing to avoid occurring the sudden change away from implementation sequence, a kind of such sudden change away from design detects in the 811a-900 sequence.It is 1 base pair disappearance, in final definite Already in 812a 1-kb box.This sudden change is not caught under the 1-kb level.It does not detect under the 10KB level, and this is because many 454 read just in time accidental the end or beginning in its vicinity coincidently, causes sudden change as read support by whole poorly, and so is ignored by automatic SNP and DIP prospecting tools.
Discuss
At this, in current research, we build 1,080,000 base pair mycoplasma mycoides genomes.But acquisition can activate to produce the faultless genome only be synthesized the new cell that genome controls be complicated and need many quality control step in recipient cell.Because the single base pair disappearance in indispensable gene dnaA failed in several weeks.In surpassing 1 megabase, the base of a mistake in indispensable gene makes the genome inactivation, and inserts and lack not impact of viability in the lots of genes group of genomic nonessential part.
During this research, we have developed many methods so that detection and the error recovery of synthetic gene group fragment.These methods comprise synthetic and combination nonsynthetic genomic fragment, follow and shift and DNA sequencing by genome, to find sequence errors and to detect the viability of synthetic design.
The successful genome of our first shifts body and uses the natural gene group of separating from intact cell.These studies have shown that genome can successfully be transferred to relevant species, the existing genetic material of displacement, and this is in the process of the phenotype that cell transformation is become to new genome defined.These experimental results show that naked DNA can be transferred.Now we ignorant be that DNA in the mycoplasma mycoides cell is methylated, and therefore when inserting the mycoplasma capri cell protected avoiding limit.
Because final synthetic gene assembling part has been recombinated by yeast, so we have developed new method for clone natural bacteria karyomit(e) (Benders etc. at yeast, Nucleic Acids Res, (on March 7th, 2010)) and separate complete natural and synthetic bacterial genomes (Lartigue etc. from yeast, Science 325,1693 (on September 25th, 2009)).Comprise the not success of initial transfer body that natural mycoplasma mycoides genomic clone is yeast centromeric plasmid.Research shows widely subsequently, need the locus specificity DNA methylation to shift for genome, unless the restriction system gene is removed (Lartigue etc., Science325,1693 (on September 25th, 2009)) from the genome of acceptor mycoplasma capri cell.
These researchs produce effective novel method, and it makes from the bacterial genomes of the species that lack genetic system grows in yeast by adding the yeast centromeric sequence.And the bacterial genomes of cloning in yeast can easily be used the yeast genetic tool---to comprise homologous recombination---and modify.The bacterial chromosome of modifying can then separate from yeast, optionally methylates, and shifts and get back to recipient cell to produce the donorcells (Lartigue etc., Science 325,1693 (on September 25th, 2009)) of modifying version.
Although the mean size lower than bacterial genomes, but we synthesize 1,077, the 947-bp double chain DNA molecule is the maximum synthetic molecules with precise structure of report, and be almost synthetic mycoplasma genitalium genome (Gibson etc. before us, Science 319,1215 (on February 29th, 2008)) twice of size.Synthetic or semi-synthetic molecule synthetic, and it has proof as connatural as the Compound Phase of natural generation, for a long time as the evidence of the molecular structure of supporting to advise.The proof that our synthetic gene group and semi-synthetic genome are given the phenotype character of mycoplasma mycoides imply its based on DNA sequence dna enough accurately to describe cell alive in detail.
The cell that the genome be introduced into (natural gene group, synthetic gene group or semi-synthetic genome) is controlled is called " synthetic cell ", even the tenuigenin of recipient cell does not synthesize.The phenotype effect of recipient cell kytoplasm is changed by protein and is copied dilution along with cell only carries the metastatic gene group.Shift on flat board and copy to form 30 divisions of colony (> or 109 times of dilutions) after, the offspring will not be included in any protein molecule existed in initial cell.The character that expection is assembled the cell that genome controls is identical, as whole cell, has been synthesized production (DNA software builds on himself hardware).
From aforementioned description, those skilled in the art can determine the fundamental characteristics of disclosure embodiment, and in the situation that do not deviate from its spirit and scope, can make a change and improve, so that it adapts to various application and condition, and to maximally utilise native system and invention.Previous embodiment is understood to it is only schematically, limits never in any form the application's scope.Above and all applications, patent of quoting in figure, the whole disclosure of open (comprising reference manual) be incorporated to this paper with its integral body by reference.
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Claims (28)

1. for generation of the method for synthetic cell, described method comprises:
(i), as one or more fragment, assemble synthetic or semisynthetic donor gene group;
(ii) described donor gene group and host's carrier are introduced to the heterologous host cell, wherein optionally described donor gene group was connected before introducing described host with described host's carrier;
(iii) reclaim described donor gene group from described host cell; With
(iv) reclaimed donor gene group is introduced to recipient cell;
Thereby generate the synthetic cell that comprises described donor gene group, wherein said donor gene group is basically complete cell, virus or organelle gene group, and it is at least minimal genome, and length is greater than about 300kb.
2. method according to claim 1, wherein said donor gene group is full cell, virus or organelle gene group basically.
3. method according to claim 1, wherein said donor gene group and described host's carrier are by simultaneously or in succession introduce described host cell.
4. method according to claim 1, wherein by described host's carrier is transformed into to the donorcells that comprises described donor gene group, be connected described host's carrier with described donor gene group, then introduces described host cell.
5. method according to claim 1, wherein said host cell is bacterial cell, fungal cell, insect cell, vegetable cell or alga cells.
6. method according to claim 1, wherein said host cell is yeast cell.
7. method according to claim 4, wherein said host's carrier is the kinetochore plasmid.
8. method according to claim 7, wherein said host's carrier is that yeast centromeric plasmid and described host cell are yeast cell.
9. method according to claim 1, wherein said host's carrier is the carrier that can be used for homologous recombination.
10. method according to claim 1, wherein said donor gene group is linearized or fracture before introducing described host cell.
11. method according to claim 1, wherein said donor gene group is bacterial genomes, fungal gene group, Yeast genome, archeobacteria genome, cyanobacteria genome, algae genome, phage genome, Mitochondrial Genome Overview, chloroplast gene group, viral genome or organelle gene group.
12. method according to claim 1, wherein host cell is eukaryotic cell or prokaryotic cell prokaryocyte.
13. method according to claim 1, also be included in described host cell and modify described donor gene group.
14. method according to claim 1, also comprise degraded or the native gene group of removing described recipient cell.
15. method according to claim 1, the donor gene group of wherein said recovery was methylated before introducing described recipient cell.
16. method according to claim 1, the restriction enzyme function of wherein said recipient cell be non-existent, be removed or inactivation.
17. method according to claim 1, wherein said recipient cell is bacterial cell, yeast cell, fungal cell, insect cell, vegetable cell or alga cells.
18. method according to claim 1, also comprise the second donor gene group is introduced to described host cell, wherein said the second donor gene group is different from described the first donor gene group, thereby produces the host cell that comprises two kinds of different donor gene groups.
19. method according to claim 18, wherein introduce described the second donor gene group and comprise the described host cell that will comprise described the first donor gene group and the second host cell mating that comprises described the second donor gene group.
20. method according to claim 1, wherein said synthetic cell is showed the phenotype corresponding with the described donor gene group that is incorporated to any modification to it.
21. method according to claim 1, wherein said donor gene group is the synthetic gene group.
22. method according to claim 1, wherein said donor gene group is semi-synthetic genome.
23. the synthetic cell produced by the described method of claim 1.
24., for the manufacture of the method for synthetic cell, described synthetic cell displaying is by synthetic donor gene group or the phenotype of semisynthetic donor gene group coding, described method comprises:
(i) described donor gene group and the host's carrier that is adapted at the described donor gene group of clone in host cell are introduced to described host cell, obtain like this product that comprises described donor gene group, described donor gene group comprises described host's carrier;
(ii) be recovered in from described host cell the described product that comprises the donor gene group obtained step (i);
(iii), the described product of (ii) is introduced to recipient cell under certain condition, so that described recipient cell is showed the phenotype by described product coding; With
(iv) reclaim the synthetic cell obtained from step (iii);
Wherein said donor gene group is basically complete cell, virus or organelle gene group, it is at least minimal genome, and the minority component that length is greater than about 300kb and comprises nucleic acid substances, described minority component shows that for described synthetic cell the phenotype by described donor gene group coding is essential.
25. method according to claim 24, further be included in described host cell and modify described donor gene group.
26. method according to claim 24, further comprise degraded or the native gene group of removing described recipient cell.
27. synthetic cell, its show by the donor gene group coding and otherwise can be by the expectation phenotype of described cell display, wherein said synthetic cell produces by the described method of claim 24.
28. synthetic cell, it comprises synthetic donor gene group or semisynthetic donor gene group, and show by described donor gene group coding and otherwise can be by the expectation phenotype of described cell display, wherein said donor gene group comprises the foreign gene group nucleic acid substances that is greater than 300kb and the genome component of showing the minimum that described expectation phenotype is essential for described synthetic cell.
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