CN107142269B - Modified plasmid replicon and application thereof - Google Patents
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- 239000013612 plasmid Substances 0.000 title claims abstract description 124
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 108020004705 Codon Proteins 0.000 claims abstract description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 4
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 4
- 239000004220 glutamic acid Substances 0.000 claims abstract description 4
- 238000010276 construction Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 2
- 101710087869 Replication protein RepA Proteins 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 19
- 101710090029 Replication-associated protein A Proteins 0.000 abstract description 8
- 230000010076 replication Effects 0.000 abstract description 6
- 150000003148 prolines Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000007788 liquid Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710150114 Protein rep Proteins 0.000 description 1
- 101710152114 Replication protein Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a modified plasmid replicon and application thereof. The plasmid replicon provided by the invention is a mutant in which 93-bit glutamic acid of RepA protein of a classical low-copy replicon pSC101 is mutated into proline; the codon encoding this proline is CCG. The plasmids carrying the artificially engineered plasmid replicon of the present invention (pSC101-RepA-E93P) extracted in vitro gave about 12-fold higher yields after replication in E.coli than the plasmids carrying the wild-type pSC101 replicon and about 2-fold higher yields than the plasmid pUC57 carrying a high copy replicon of the CorEI/pMB1/pBR322 type. Meanwhile, in E.coli, a plasmid carrying the artificially modified plasmid replicon of the present invention can co-replicate almost equally in proportion to a plasmid carrying a CorEI/pMB1/pBR322 type high copy replicon.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a modified plasmid replicon and application thereof.
Technical Field
Plasmids are small DNA molecules that are capable of autonomous replication independently of the chromosomes in a cell. It is a functional carrier necessary for engineering modification of animals, plants and bacteria. The plasmid can carry exogenous or artificially synthesized genes and enter animal, plant and bacterial cells, thereby realizing the functional modification of host cells. The plasmid has important application value in the fields of molecular biology, genetic engineering modification, synthetic biology and DNA treatment.
The replication of plasmids in host cells requires the dependence on a special DNA element: a plasmid replicon. Types of currently common E.coli plasmid replicons include pSC101, p15A, R6K, CorEI/pMB1/pBR322, and the like. Plasmids with different types of replicons usually possess different copies, varying in number from a few to thousands. This difference in plasmid copy number allows for the extraction of plasmids with different replicons in vitro with greatly different yields after replication in a host cell. In order to be able to prepare plasmid DNA in large quantities in vitro, most plasmids used for functional engineering of animal, plant, microbial cells or related applications will utilize plasmid replicons that are capable of producing relatively high copies as replication elements. Currently, the replicon type with the highest copy number is CorEI/pMB1/pBR322 type replicon, and 90% of high-copy plasmids use this type of replicon, such as classical vectors pUC57, pCDNA3.1 and the like. Plasmids carrying replicons of this type are often extracted several to several tens times higher in vitro than plasmids carrying low copy Plasmid replicons such as pSC101 type (. about.5 copies) (references: Wang et al 2009, Plasmid; Balb. s. et al 1996, Gene; Summers et al 1985, Bioessays).
Since the high-copy replicons found so far are so few that the construction of most of the high-copy plasmids can only use the plasmid replicons of the CorEI/pMB1/pBR322 type, which greatly limits the diversity of plasmid construction, it is of great interest to find new high-copy replicons. Meanwhile, plasmids with high copy replicons of the same type cannot exist in one cell at the same time, so that two high copy plasmids with CorEI/pMB1/pBR322 replicons cannot be used for expressing different genes in the same Escherichia coli cell at the same time. Therefore, in order to simultaneously use two different high-copy plasmids for the co-expression study of foreign genes in E.coli, it is particularly necessary to develop a high-copy replicon of a type different from CorEI/pMB1/pBR 322.
Disclosure of Invention
The invention aims to solve the technical problem of providing an artificially modified plasmid replicon and application thereof.
The purpose of the invention can be realized by the following technical scheme:
an artificially modified plasmid replicon pSC101-RepA-E93P is obtained by mutating glutamic acid at position 93 of RepA of a pSC101 replication protein into proline. The sequence of the wild-type pSC101 replicon is shown in SEQ ID NO. 1.
The codon of the proline in the artificially modified plasmid replicon pSC101-RepA-E93P is preferably CCG.
A further preferable sequence of the artificially modified plasmid replicon pSC101-RepA-E93P is shown in SEQ ID NO. 2.
A plasmid comprising the plasmid replicon pSC101-RepA-E93P of claim 1 or 2.
The plasmid is preferably pGENT1 containing the plasmid replicon pSC 101-RepA-E93P.
The invention relates to the application of an artificially modified plasmid replicon pSC101-RepA-E93P in plasmid construction; preferably in the construction of high copy plasmids.
The invention relates to application of an artificially modified plasmid replicon pSC101-RepA-E93P in improving the yield of plasmid in vitro extraction.
Advantageous effects
The invention is based on a classical low-copy plasmid replicon pSC101, and obtains a novel high-copy plasmid replicon pSC101-RepA-E93P through mutation transformation. The plasmid replicon can be conveniently applied to the construction of high-copy plasmids; plasmids carrying the pSC101-RepA-E93P high copy Plasmid replicon gave, after replication in E.coli, yields of about 12 times as high as plasmids carrying the wild-type pSC101 replicon, about 2 times as high as Plasmid pUC57 carrying a high copy replicon of the CorEI/pMB1/pBR322 type, and about 8 times as high as plasmids carrying the reported pSC101-RepA-E93K and pSC101-RepA-E93R high copy replicons (Peterson J and Phillips GJ,2008, Plasmid.) (example 1). Meanwhile, in Escherichia coli, the plasmid can be replicated together with the reported plasmid carrying the CorEI/pMB1/pBR322 type high-copy replicon in almost the same proportion, and a foundation is laid for solving the problem that the high-copy plasmids of the replicon of the same type are incompatible and realizing the functional research in Escherichia coli cells by using different high-copy plasmids. In conclusion, the high-copy replicon obtained by artificial modification is a novel high-copy replicon which is not reported before, has strong applicability and can effectively solve the problem of plasmid incompatibility in the cotransformation of different high-copy plasmids with the CorEI/pMB1/pBR322 replicon at present.
Description of the drawings:
FIG. 1 is a schematic structural diagram of an artificially engineered plasmid replicon pSC 101-RepA-E93P.
FIG. 2 is a map of plasmid pGENT1 carrying the wild-type pSC101 replicon.
FIG. 3 is a schematic representation of plasmid pGENTS (RepA-E93P) carrying the pSC101-RepA-E93P replicon.
FIG. 4 is a linearized plasmid electrophoretogram of extracted pGENT1(pSC101), pGENTS (pSC101-RepA-E93P), pUC57(CorEI/pMB1/pBR322), pGENT2(pSC101-RepA-E93K) and pGENT3(pSC 101-RepA-E93R).
FIG. 5 is a result of analyzing the yields of the co-transformed pGENTS and pUC57 in E.coli by Bioanalyzer.
The specific implementation mode is as follows:
example 1: in vitro engineering screening of pSC101-RepA-E93P high copy replicons
The objective of this experiment was to obtain a high copy number of the pSC101-RepA-E93P mutant. The method comprises the following steps:
1) in the reported wild-type pSC101 replicon (Cohen et al 1973 Proc Natl Acad Sci US A; cohen et al.1977J Bacteriol; based on the Vocke C and Bastia D.1983Proc Natl Acad SciUSA sequence (SEQ ID NO.1), a plasmid pGENT1 (carrying pSC101-RepA wild type replicon, a selection marker for Kan resistance and LacZ) with the wild type replicon was synthesized, and the map is shown in FIG. 2; the sequence is shown in SEQ ID NO.3), designing a primer aiming at the saturation mutation at the E93 position of RepA protein, and obtaining the primerThe middle F primer is: gtccactggaaaatnnnaaagcctttaaccaaaggattcctgatt, respectively; the primer R is as follows: attttccagtggacaaactatgccaagttctcaagcgaaaaatta, wherein n is degenerate nucleotide, selected from any one of a, c, t and g. The primers were synthesized by GenScript.
2) A library of random saturated mutants of the pSC101 replicon RepA protein E93 was constructed using a PCR amplification mutagenesis method. Wherein, the PCR system is as follows: DNA polymerase: 0.5 mul; 10 × PBO buffer: 5 mu l of the solution; dNTP: 0.7 μ l; BSA 0.9. mu.l; f primer: 0.2 ul; r primer: 0.2 ul; template plasmid: 0.5 ul. The PCR procedure was: 95 ℃ for 5 min; 25 cycles of 95 ℃, 30s- >55 ℃, 30s- >72 ℃ and 4 min; 72 ℃ for 7 min.
3) The PCR amplified library of pGENT1 plasmid (pGENT 1-E93N for short) carrying random mutation at RepA-E93 position is recovered by tapping DNA gel recovery kit (Axygen, USA).
4) The PCR amplification library obtained in step 3 was transformed into E.coli Top10 strain, plated on LB (Kan) -resistant plates, and cultured overnight at 37 ℃. Meanwhile, the wild-type plasmid pGENT1 was transformed into E.coli Top10 strain, which was coated with LB (Kan) resistant plate and LB (Amp) resistant plate, respectively, and cultured overnight at 37 ℃ as the control for the next experiment.
5) 96 single clones were picked from plates transformed with pGENT1-E93N mutant library, inoculated into 96 tubes of LB liquid medium, and cultured overnight at 37 ℃. Meanwhile, single clones were picked from plates transformed with wild-type pGENT1, inoculated into LB liquid medium, and cultured overnight at 37 ℃.
6) From the culture obtained in step 5, 1ml of each of the bacteria (OD) was collected600The balance is to-1.5, the bacteria quantity is ensured to be the same), and plasmids are extracted by utilizing Axygen (USA) plasmid miniextraction kit. The mutants with the highest plasmid yield (pGENTS for short) were screened using the plasmids quantitatively extracted by NanoDrop.
7) The mutant pGENTS with the highest yield obtained by screening is transformed into escherichia coli Top10 again, and the stability of the yield is verified; pGENT1, a high copy plasmid pUC57 carrying a CorEI/pMB1/pBR322 type replicon, was simultaneously transformed as a control; LB plates corresponding to the resistance were spread separately and cultured overnight at 37 ℃. In view of the reported literature that the mutation of 93E of the RepA protein of the pSC101 replicon into K and R can significantly improve the copy number of the Plasmid (Peterson J and Phillips GJ,2008, Plasmid.), the mutants pGENT2 (the RepA of the pSC101 replicon region, and the mutation of E93 into K) and pGENT3 (the RepA of the pSC101 replicon region, and the mutation of E93 into R) of the Plasmid pGENT1 are constructed at the same time to be used as experimental controls, and the Plasmid pGENT1 and the Plasmid pGENT2 are respectively transformed into Escherichia coli Top10, coated with a resistant plate, and cultured overnight at 37 ℃.
8) Single colonies were picked from plates transformed with pGENT1, pGENTS, pUC57, pGENT2 and pGENT3 plasmids, inoculated into the corresponding resistant LB liquid medium, and cultured overnight at 37 ℃.
9) From the culture obtained in step 8, 1ml of each of the bacteria (OD) was collected600Balanced to-1.5, ensuring the same bacterial load), pGENT1, pGENTS, pUC57, pGENT2 and pGENT3 plasmids were extracted respectively with Axygen (USA) plasmid miniprep kit, the plasmids quantitatively extracted with NanoDrop, 50ul H2And O, eluting the plasmid. Meanwhile, EcoRI is used for cutting linearized plasmids, agarose gel electrophoresis is used for preliminarily observing the difference of plasmid yield, and meanwhile, the extracted pGENTS plasmids are sent for sequencing.
The results are as follows: as shown in Table 1, the pGENTS plasmids obtained by the screening were produced in 12.6 times as much as the wild-type plasmid pGENT1 and in 2.3 times as much as the high-copy plasmid pUC57 which is currently used. Meanwhile, the pGENTS Plasmid yield was about 8 times that of Plasmid pGENT2 carrying the literature reported pSC101 high copy replicon mutant pSC101-RepA-E93K (Peterson J and Phillips GJ,2008, Plasmid.) and of Plasmid pGENT3 carrying the literature reported pSC101 high copy replicon mutant pSC101-RepA-E93R (Peterson J and Phillips GJ,2008, Plasmid.) (Table 1). Sequencing finds that the amino acid at position 93 of the pSC101 replicon RepA protein carried by pGENTS is mutated from the original glutamic acid (E) into proline (P), and the codon for the proline is CCG. Agarose electrophoresis results also showed that extracted pGENTS (pSC101-RepA-E93P) plasmids possessed higher concentrations than pGENT1(pSC101), pUC57(CorEI/pMB1/pBR322), pGENT2(pSC101-RepA-E93K) and pGENT3(pSC101-RepA-E93R) plasmids (FIG. 4).
TABLE 1 comparison of plasmid yields for pGENT1, pGENTS, pUC57, pGENT2 and pGENT3
| Plasmids | Amount of plasmid (ng/ul) |
| pGENT1 | 15.84±1.4 |
| pGENTS | 199.8±3.5 |
| pUC57 | 86.76±2.7 |
| pGENT2 | 25.78±3.0 |
| pGENT3 | 23.56±2.2 |
Example 2: co-transformation of plasmid pGENTS carrying the pSC101-RepA-E93P high copy replicon with plasmid pUC57 carrying the CorEI/pMB1/pBR322 high copy replicon
1) pGENTS and pUC57 were transformed into E.coli Top10 simultaneously, plated with LB (Kan-resistant + Amp-resistant) plates, and cultured overnight at 37 ℃.
2) From the transformed plate of E.coli co-transformed with pGENTS and pUC57 plasmids, a single clone was selected, inoculated into LB liquid medium, and cultured overnight at 37 ℃.
3) From the culture of step 2), 1ml of the bacteria (OD) were taken600The balance is to-1.5, the bacteria quantity is ensured to be the same), and plasmids are extracted by utilizing Axygen (USA) plasmid miniextraction kit.
4) The plasmid obtained in step 3) was linearized by digestion with EcoRI and the proportions of pUC57 and pGENTS in E.coli were analysed by an Agilent 2100 bioanalyzer.
As a result: as shown in FIG. 5, both pGENTS and pUC57 can be present in E.coli in comparable amounts. Bioanalyzer analysis showed that pGENTS was produced in E.coli 1.3 times as much as pUC57 and 0.9 times as many molecules as pUC 57. pGENTS and pUC57 exist in Escherichia coli almost equally, and provide a good foundation for functional research and application of the two types of high-copy plasmids.
<110> Nanjing Kinsrui Biotechnology Ltd
<120> a modified plasmid replicon and applications thereof
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agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 120
gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg ctgccttact 180
gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt cagactggaa 240
aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat agcagacccg 300
ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt agtttccttg 360
catgaatcca taaaaggcgc ctgtagtgcc atttaccccc attcactgcc agagccgtga 420
gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag gtgcctgatg gtcggagaca 480
aaaggaatat tcagcgattt gcccgagctt gcgagggtgc tacttaagcc tttagggttt 540
taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta atactgcgga 600
actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat ctttttttat 660
tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac acacaaaggt 720
ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa ggtctagcag 780
aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg actagcccat 840
ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc tgaattagtt900
gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca tgaaaccaag 960
ctaattttat gctgtgtggc actactcaac cccacgattg aaaaccctac aaggaaagaa 1020
cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag tagggaaaat 1080
gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt ggaaatcagg 1140
aatcctttgg ttaaaggctt tgagattttc cagtggacaa actatgccaa gttctcaagc 1200
gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca gttaaaaaaa 1260
ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc tatgaggatt 1320
tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa tatagagatt 1380
agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga gtttaaaagg 1440
cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag caatatgaaa 1500
ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt tgaactagat 1560
agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa 1620
ataccaacaa ccattacatc agattcctac ctacgtaacg gactaagaaa aacactacac 1680
gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt gagtgacatg 1740
caaagtaagc atgatctcaa tggttcgttc tcatggctca cgcaaaaaca acgaaccaca 1800
ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc ttgtatctat 1860
cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt agatatcaaa 1920
gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata catcagagct 1980
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ctgtcagacc aagtttacga gctcgcttgg actcctgttg atagatccag taatgacctc 60
agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 120
gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg ctgccttact 180
gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt cagactggaa 240
aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat agcagacccg 300
ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt agtttccttg 360
catgaatcca taaaaggcgc ctgtagtgcc atttaccccc attcactgcc agagccgtga 420
gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag gtgcctgatg gtcggagaca 480
aaaggaatat tcagcgattt gcccgagctt gcgagggtgc tacttaagcc tttagggttt 540
taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta atactgcgga 600
actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat ctttttttat 660
tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac acacaaaggt 720
ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa ggtctagcag 780
aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg actagcccat 840
ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc tgaattagtt 900
gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca tgaaaccaag 960
ctaattttat gctgtgtggc actactcaac cccacgattg aaaaccctac aaggaaagaa 1020
cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag tagggaaaat 1080
gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt ggaaatcagg 1140
aatcctttgg ttaaaggctt tccgattttc cagtggacaa actatgccaa gttctcaagc 1200
gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca gttaaaaaaa 1260
ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc tatgaggatt 1320
tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa tatagagatt 1380
agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga gtttaaaagg 1440
cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag caatatgaaa 1500
ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt tgaactagat 1560
agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa 1620
ataccaacaa ccattacatc agattcctac ctacgtaacg gactaagaaa aacactacac 1680
gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt gagtgacatg 1740
caaagtaagc atgatctcaa tggttcgttc tcatggctca cgcaaaaaca acgaaccaca 1800
ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc ttgtatctat 1860
cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt agatatcaaa 1920
gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata catcagagct 1980
tttacgagtt tttggtgcat ttaaagctgt tcaccatgaa cagatcgaca atgtaac 2037
<210>3
<211>4027
<212>DNA
<213> Artificial sequence
<220>
<223> pGENT1 plasmid sequence
<400>3
gttacattgt cgatctgttc atggtgaaca gctttaaatg caccaaaaac tcgtaaaagc 60
tctgatgtat ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt 120
gatatctaac ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata 180
gatacaagag ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt 240
ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatgct tactttgcat 300
gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg 360
tagtgttttt cttagtccgt tacgtaggta ggaatctgat gtaatggttg ttggtatttt 420
gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc 480
tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt 540
catattgctg taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct 600
tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat 660
ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat 720
cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt 780
ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct 840
tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct 900
gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt 960
ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc 1020
tttccttgta gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt 1080
ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac 1140
taattcagac atacatctca attggtctag gtgattttaa tcactatacc aattgagatg 1200
ggctagtcaa tgataattac atgtcctttt cctttgagtt gtgggtatct gtaaattctg 1260
ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc 1320
tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata 1380
aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc 1440
gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa 1500
ccctaaaggc ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt 1560
ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca 1620
cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa 1680
ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg 1740
gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc 1800
cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt 1860
aaggcagcgg tatcatcaac aggcttaccc gtcttactgt ccctagtgct tggattctca 1920
ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag 1980
gtcattactg gatctatcaa caggagtcca agcgagctcg taaacttggt ctgacaggaa 2040
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2100
attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2160
agttttaaat caagcccaat ctgaataatg ttacaaccaa ttaaccaatt ctgattagaa 2220
aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata 2280
tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat 2340
ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa 2400
tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc 2460
cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt 2520
acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg 2580
agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa 2640
ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc 2700
taatacctgg aatgctgttt ttccggggat cgcagtggtg agtaaccatg catcatcagg 2760
agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct 2820
gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc 2880
tggcgcatcg ggcttcccat acaagcgata gattgtcgca cctgattgcc cgacattatc 2940
gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga 3000
cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag 3060
ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga 3120
cacgggccag agctgcatcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 3180
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 3240
gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 3300
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 3360
ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 3420
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 3480
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gagaattcga 3540
gctcggtacc tcgcgaatac atctagatat cggatcccgg gcccgtcgac tgcagaggcc 3600
tgcatgcaag cttggtgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 3660
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 3720
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 3780
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 3840
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3900
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3960
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4020
Claims (6)
1. An artificially-modified plasmid replicon, which is obtained by mutating glutamic acid at position 93 of a pSC101 replication protein RepA into proline.
2. The artificially engineered plasmid replicon according to claim 1, wherein the codon encoding the proline is CCG; the plasmid replicon sequence is shown in SEQ ID NO. 2.
3. A plasmid comprising the plasmid replicon of claim 1 or 2.
4. Use of the artificially engineered plasmid replicon of claim 1 or 2 for plasmid construction.
5. Use according to claim 4, characterized in that it is the use of the artificially engineered plasmid replicon of claim 1 or 2 for constructing high copy plasmids.
6. Use of the artificially engineered plasmid replicon of claim 1 or 2 for increasing the yield of plasmid in vitro extraction.
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| CN101302531A (en) * | 2008-06-20 | 2008-11-12 | 南京师范大学 | A kind of BAC vector and its construction method for shuttle expression between Escherichia coli-Streptomyces-Pseudomonas |
| CN101633901A (en) * | 2009-08-14 | 2010-01-27 | 南京师范大学 | Escherichia coli strain for recombined engineering |
| CN103525861A (en) * | 2013-05-07 | 2014-01-22 | 江苏省农业科学院 | Binary Ti plasmid with bidirectional promoter trapping and plasmid rescue functions and its construction method |
| CN104480130A (en) * | 2014-11-20 | 2015-04-01 | 苏州金唯智生物科技有限公司 | PTerm-SC plasmid as well as construction method and application thereof |
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| US5919678A (en) * | 1992-03-13 | 1999-07-06 | Institut National De La Recherche Agronomique | Methods for using a temperature-sensitive plasmid |
| CN101302531A (en) * | 2008-06-20 | 2008-11-12 | 南京师范大学 | A kind of BAC vector and its construction method for shuttle expression between Escherichia coli-Streptomyces-Pseudomonas |
| CN101633901A (en) * | 2009-08-14 | 2010-01-27 | 南京师范大学 | Escherichia coli strain for recombined engineering |
| CN103525861A (en) * | 2013-05-07 | 2014-01-22 | 江苏省农业科学院 | Binary Ti plasmid with bidirectional promoter trapping and plasmid rescue functions and its construction method |
| CN104480130A (en) * | 2014-11-20 | 2015-04-01 | 苏州金唯智生物科技有限公司 | PTerm-SC plasmid as well as construction method and application thereof |
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| New pSC101-derivative cloning vectors with elevated copy numbers;James Peterson 等;《Plasmid》;20080531;第59卷(第3期);摘要，第197-198页第3.1-3.3节，Table 2 * |
| Synthetic construct Rep101 (rep101) gene, complete cds;Shetty,R.P. 等;《GenBank》;20080421;EU496096.1 * |
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