CN109943581B - A plasmid and phage-assisted continuous directed evolution system and directed evolution method - Google Patents

Abstract

The invention relates to the field of directed evolution of membrane proteins, in particular to a plasmid and phage-assisted continuous directed evolution system and a directed evolution method. A plasmid, comprising one or both of an AP1 plasmid and an AP2 plasmid; the AP1 plasmid carries gIII gene, and a functional protein recognition site is arranged between a promoter and a ribosome binding site of the plasmid; the AP2 plasmid carries a functional protein gene. The invention designs new auxiliary plasmids AP1 and AP2, couples the transport activity of target protein to intracellular and extracellular substrate molecules with gIII expression on AP1, couples the reproductive capacity of SP with the activity of the target protein in an indirect mode, and achieves the effect of continuously and directionally evolving the target protein.

Description

A Plasmid and Phage-Assisted Continuous Directed Evolution System and Directed Evolution method

technical field

The invention relates to the field of membrane protein directed evolution, in particular to a plasmid and phage-assisted continuous directed evolution system and directed evolution method.

Background technique

In the genome, about 30% of the gene products are membrane proteins, which shows the importance of membrane proteins in organisms. Membrane proteins mainly include signal receptors, transporters, ion channel proteins and some enzymes, which are crucial to cell metabolism, physiological balance and intracellular regulation. In the process of drug development and design, many membrane proteins are the targets of drug design. However, the lack of membrane protein structure and biochemical information restricts the development of drug design, because the instability and insolubility of membrane proteins make it difficult for researchers to obtain high-purity Analyze and study the three-dimensional structure of the membrane protein. For these properties of membrane proteins, directed evolution may be a powerful tool to help researchers understand the relationship between membrane protein structure and its biological function.

Traditional methods of mutagenesis and genetic recombination to evolve membrane proteins are time-consuming and labor-intensive, requiring the construction of mutation libraries and repeated screening (Journal of Theoretical Biology, 2000, 205(3): 483-503). The current relatively novel methods such as liposome display are complicated to operate, and an expensive in vitro translation system needs to be introduced, which is costly (Biophysics, 2015, 11:67-72).

The team of David R Liu at Harvard University invented a phage-assisted continuous evolution system (PACE). The system mainly consists of three parts. The first part puts the gene to be evolved on the phage M13 genome and replaces the original gIII gene to form SP. SP lacks gIII and cannot infect the host and produce progeny phage; the second part puts SP The gIII gene required for infecting and producing progeny phages is placed on an additional plasmid for expression to form an auxiliary plasmid AP. The expression of gIII is regulated by the activity of the target gene on the SP, that is, the reproductive ability of the SP in the cell and the target gene. The biological activities are coupled together; the third part is the mutagenesis plasmid MP, which uses arabinose inducer to induce the expression of genes DNAQ926, dam and seqA on the MP, so that the wrong base cannot be repaired during the DNA replication process, and the mutation rate is increased by several hundred times. When the SP with the target gene infects the host, the SP replicates the genetic material in the host cell, and with the help of MP, various mutants of the SP will be produced. If the target gene on the SP is positively mutated, the gIII protein on the AP plasmid is expressed, which can package the invasive progeny SP phage and secrete it out of the cell, and enter the next round of infection, mutation, and progeny. Circulation; if the target gene on SP is not mutated or negatively mutated, the gIII protein on the AP plasmid will not be expressed, and the progeny produced will not be secreted outside the cell or the number will be small; the evolution pool is continuously diluted at a certain flow rate, and the positive mutation The SP can continue to produce offspring and remain in the pool, and the negatively mutated SP will continue to flow out of the pool and disappear. David R Liu's team successfully evolved T7 RNA polymerase (NATURE.2011April; 472:498–505), protease (Nature Communications, 2014, 5:5352.), DNA binding protein (Nature Methods, 2015, 12 (10 ):939.) etc. and endowed these proteins with new properties, but did not involve the field of membrane proteins. The existing PACE system is not suitable for directed evolution of membrane proteins.

In view of this, the present invention is proposed.

Contents of the invention

The present invention designs new auxiliary plasmids AP1 and AP2, the protein expressed by the functional gene on AP2 inhibits the expression of gIII protein on AP1, the transporter transports the substrate into the cell, and the substrate molecule can release the inhibitory effect of the functional protein, which is about to be transported The transport activity of the protein on extracellular substrate molecules is coupled with the expression of gIII on AP1, and in this indirect way, the reproductive ability of the phage is coupled with the transport activity of the transport protein to achieve the effect of continuous directed evolution of the target protein.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

A plasmid, including one or both of the AP1 plasmid and the AP2 plasmid;

The AP1 plasmid carries the gIII gene, and a functional protein recognition site is set between the plasmid promoter and the ribosome binding site;

The AP2 plasmid carries a functional protein gene.

The AP1 plasmid provided by the present invention is an auxiliary plasmid for the expression of gIII, an essential gene for phage infection and proliferation; the AP1 plasmid carries the gIII gene and is activated by a promoter such as the J23109 promoter, and there is a recognition site for a functional protein between the promoter and RBS, In the absence of intracellular inducers, gIII expression is suppressed. The AP2 plasmid is a helper plasmid that indirectly couples phage proliferation ability to the concentration of extracellular substrate molecules and the activity of membrane proteins such as transporters.

Further, the promoter is J23109 promoter.

Preferably, the functional protein recognition site in the AP1 is recognized by the functional protein encoded by the functional protein gene of the AP2 plasmid, which acts as a repressor.

That is to say, the functional protein encoded by the functional protein gene of the AP2 plasmid is used to combine with the functional protein recognition site in the AP1 plasmid, thereby achieving the repression effect.

The functional protein set in the present invention is used to respond to the membrane protein substrate molecule transported into the cell, release the repression effect, and induce the expression of the gIII gene.

In the present invention, different functional proteins correspond to different substrate molecules, and different substrate molecules are used to evolve different target genes, such as the target gene is the lactose transporter gene, the substrate molecule is a disaccharide, and the functional protein is the functional protein CelR .

Further, the nucleic acid sequence of the functional protein CelR recognition site is shown in SEQ ID NO.3 (14bp).

Further, the gene sequence of the AP1 plasmid is shown in SEQ ID NO.1, and the gene sequence of the AP2 plasmid is shown in SEQ ID NO.2.

The present invention also provides a phage-assisted continuous directed evolution system, which contains the above-mentioned AP1 plasmid and AP2 plasmid.

Further, the continuous directed evolution system also includes phages, host bacteria, and mutagenic plasmids for replacing the gIII gene with the target gene.

Further, the AP1 plasmid and AP2 plasmid exist in the form of being transferred into the host bacteria.

Further, the target gene includes a gene of a membrane protein;

Preferably, the membrane proteins include transporter proteins, receptor proteins, and ion channel proteins.

Further, the host bacteria is Escherichia coli carrying F factor.

The host bacterium is preferably E.coli S1030.

The present invention also provides a directed evolution method using the above-mentioned phage-assisted continuous directed evolution system, wherein the AP1 plasmid, the AP2 plasmid and the mutagenic plasmid are transferred into the host bacteria to obtain the evolved host bacteria;

The evolutionary host bacteria and the phage whose gIII gene is replaced by the target gene are subjected to multiple rounds of culture and screening under the condition that the screening pressure gradually increases to obtain mutants.

The evolution method provided by the invention realizes directed evolution of membrane proteins and provides important technical support for the evolution of membrane proteins.

Further, the target gene is the lactose transporter gene, and the

The screening pressure is carried out by controlling the concentration of the extracellular substrate cellobiose, and the screening pressure is gradually increased: the concentration of the extracellular substrate cellobiose is gradually reduced from 29 mM to below 29 μM.

The speed of gradual reduction can be: reduce in the ratio of 2-15 times, such as 2 times the speed, 4 times the speed, 6 times the speed, 8 times the speed, 10 times the speed, 12 times the speed, 15 times the speed and so on.

In this method, in each round of multiple rounds of culture, 1/10 of the volume of the supernatant from the previous round can be used as the starting phage for the next round, and fresh evolutionary host bacteria are replaced in each round to ensure that mutations are accumulated on the target gene on the phage; Each round of culture induces phage proliferation by adding membrane proteins or substrate molecules for transporters. The multi-round culture and screening process of the present invention is to increase the screening pressure by continuously reducing the molecular concentration of substrates such as membrane proteins or transport proteins, so that the evolution direction is oriented to improve the transfer efficiency of the target gene to the substrate molecule, so as to realize the evolution of the target gene .

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention designs new helper plasmids AP1 and AP2, and couples the transport activity of the target protein to extracellular and extracellular substrate molecules with the expression of gIII on AP1, and in this indirect way the reproductive ability of the phage is linked to the target protein. Active coupling achieves the effect of continuous directed evolution of the target protein.

(2) The phage-assisted continuous directed evolution system provided by the present invention has been verified to be effective for the continuous directed evolution of the target protein.

(3) The phage-assisted continuous directed evolution system provided by the present invention can be used for the directed evolution of various membrane proteins such as transporter proteins, receptor proteins, ion channel proteins and the like.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Fig. 1 is a schematic diagram of phage-assisted continuous directed evolution of cellobiose, taking lacY as an example provided by the embodiment of the present invention;

2 is a schematic diagram of the gene map of the helper plasmid AP1 in Example 1 of the present invention;

3 is a schematic diagram of the gene map of the helper plasmid AP2 in Example 1 of the present invention;

4 is a schematic diagram of the gene map of the mutagenized plasmid MP in Example 1 of the present invention;

5 is a schematic diagram of the gene map of phage SP-lacY in Example 1 of the present invention;

Figure 6 is a linear relationship diagram between the phage proliferation rate and the extracellular cellobiose concentration in Example 2 of the present invention;

7 is a schematic diagram of the identification of LacY mutation sites in phage-assisted continuous directed evolution phylogenetic evolution in Example 3 of the present invention;

Fig. 8 is a graph showing the linear relationship between the transport activity of the mutant obtained in Example 4 of the present invention and the control group.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The existing designed PACE system cannot be used for directed evolution of membrane proteins, especially transport proteins. The phage-assisted continuous directed evolution system provided by the present invention comprises phage SP carrying a quasi-evolutionary transporter gene, an arabinose-induced mutant plasmid MP, and an auxiliary plasmid AP1 expressing gIII, an essential gene for SP infection and proliferation, to regulate host phage infection Resistance and helper plasmid AP2 that indirectly couples SP proliferative capacity to extracellular substrate molecule concentration and transporter activity. The invention can be used for directed evolution of various membrane proteins and transport proteins.

The present invention takes the lactose transporter LacY as an example to illustrate that the transport activity of LacY on cellobiose is improved through PACE evolution, but the protection of the present invention is not limited to the evolution of the transporter LacY and its activity on cellobiose, such as the evolution of LacY on cellobiose Evolution of transport activity of other sugars, evolution of transport activity of other transporters or ion channel proteins on their substrates, etc.

Taking LacY as an example, the schematic diagram of its cellobiose transport activity evolution using the improved PACE system is shown in Figure 1.

SP carries the target gene lacY to be evolved. AP1 (SEQ ID NO.1) carries the gIII gene and is driven by the J23109 promoter. There is a recognition site for the functional protein CelR between the promoter and RBS. In the absence of cellobiose in the cell, the expression of gIII is inhibited. AP2 (SEQ ID NO.2) carries the CelR protein gene. Both the mutagenized plasmid MP and the host strain S1030 were provided by the laboratory of David R Liu, and their genetic information has been reported in relevant literature (Nat Chem Biol.2014March; 10(3):216-222).

The host bacteria in the present invention are not limited to E.coli S1030, as long as Escherichia coli carrying F factor is acceptable.

Package wild-type SP-lacY (SEQ ID NO.3) Transform S1030-AP1/AP2 competent cells with SP carrying lacY, recover for 2 hours and add 1% cellobiose, mix the recovered bacteria with soft agar and spread evenly on plates containing solid agar. Overnight at 37°C, observe the phage plaques and take a single phage plaque on the plate and place it in 5 mL logarithmic phase (OD ₆₀₀ =0.4) S1030-AP1/AP2/MP host bacteria, add cellobiose at a final concentration of 1%, 37 Cultivate at 150rpm for 6h. The culture solution was centrifuged, and the supernatant was filtered through a 0.22 μM filter membrane to obtain the wild-type SP-lacY phage, and the titer of the wild-type SP-lacY phage was verified. The relationship between cellobiose concentration and phage SP-lacY propagation speed was tested. Only when SP-lacY can respond to the extracellular cellobiose concentration, can the cellobiose concentration be continuously reduced during the evolution of PACE, and the screening pressure can be increased, so that the evolution can move toward Increased lacY proceeds in the direction of cellobiose transport activity. PACE evolved LacY, the initial phage was wild-type SP-lacY, the titer was 1×10 ⁵ pfu/mL, the host was S1030-AP1/AP2/MP (OD ₆₀₀ =0.4), the evolution system was 1mL, and the arabinose was always maintained at 1%. The concentration of cellobiose in the first round was 1%; in the second round, 100 μl of the supernatant from the first round was used as the starting phage, and replaced with fresh S1030-AP1/AP2/MP (OD ₆₀₀ =0.4) to concentrate the mutation on the phage, and the cellobiose The sugar concentration was reduced to 0.5%, and so on, each round was diluted 10 times, and the fresh host was replaced in each round, the cellobiose was gradually reduced, and the evolution time of each round was 1h in the early stage and 2h in the late stage. Each round was sampled to detect the phage titer and sequenced to detect the LacY mutation.

The embodiment of the present invention is illustrated by taking the lactose transporter LacY as an example.

Example 1

Packaging of bacteriophage SP-lacY carrying quasi-evolved genes

1) Construction of the gIII protein expression plasmid AP1: the gIII protein is promoted by the phage shock promoter J23109, and the nucleic acid sequence (SEQ ID NO.3) of the recognition site of the CelR protein is inserted downstream of the promoter. The map of AP1 is shown in Figure 2.

2) Construction of expression plasmid AP2 for functional protein CelR protein: CelR is expressed by a constitutive promoter ( FIG. 3 ).

3) The mutagenic plasmid MP was donated by David R Liu's laboratory, and the map is shown in Figure 4.

4) AP1, AP2 and MP co-transform S1030 competent cells to obtain host S1030-AP1/AP2/MP. Before the host does not have LacY protein to transport cellobiose into the cell, the CelR protein expressed on AP2 and the CelR protein on AP1 The CelR recognition site binds to repress the expression of the downstream gene gIII; MP is a plasmid that improves mutations and is induced by arabinose. Its gene sequence is the same as that of the plasmid IP in the application number 201610349254.3.

5) PCR amplifies the M13 phage large frame SP (excluding the gIII gene), PCR amplifies the wild-type lacY gene, gibson connects the M13 phage large frame SP and the lacY gene to form an SP-lacY double-stranded plasmid, and the ligated product is used for future use. The gene map of SP-lacY is shown in FIG. 5 .

6) In step 5) the ligation product SP-lacY is transformed into host S1030-AP1/AP2/MP competent cells, recovered for 2 hours and added with a final concentration of 1% cellobiose, during the recovery stage LacY transports cellobiose into the cell, and the cells Inner cellobiose relieves the inhibitory effect of CelR protein on the gIII protein on AP1, gIII is expressed, the phage is packaged in the cell and secretes the progeny SP-lacY phage with the ability to infect, and the revived bacteria are mixed with soft agar and spread evenly on plates containing solid agar.

7) Overnight at 37°C, take a single phage plaque on the plate and place it in 5 mL of logarithmic phase (OD ₆₀₀ =0.4) S1030-AP1/AP2/MP host bacteria, add cellobiose at a final concentration of 1%, at 37°C, Cultivate at 150rpm for 6h. The supernatant of the culture solution was centrifuged and filtered through a 0.22 μm filter membrane to obtain the wild-type SP-lacY phage, and the titer of the wild-type SP-lacY phage was verified to be 1×10 ¹¹ pfu/mL.

In the present invention, plasmids AP1, AP2, MP and double-stranded SP-lacY bacteria can be obtained by conventional molecular cloning methods such as PCR, enzyme-cut ligation, and gene recombination with reference to gene maps and sequences. These molecular cloning techniques are well known in the art , the corresponding Escherichia coli, phage, and template can be obtained exactly. Therefore, the host bacteria, plasmids and phages of the present invention have the characteristics of reproducibility, and those skilled in the art can obtain them according to conventional methods.

Example 2

Verification of the relationship between the concentration of cellobiose and the propagation speed of bacteriophage SP-lacY

1) The host S1030-AP1/AP2/MP LB medium was cultured to the logarithmic phase (OD ₆₀₀ =0.4).

2) Dilute the wild-type SP-lacY phage and add it to the above logarithmic phase host to make the initial concentration in the system 50pfu/mL.

3) The final concentration gradient of cellobiose is 29mM, 14.5mM, 2.9mM, 1.45mM, 0.29mM, 0.0029mM, 0.00029mM, 0.00mM.

4) Samples were taken every 15 minutes for each cellobiose gradient, and the proliferation of phage SP-lacY in the system was detected, and the results are shown in FIG. 6 .

In Fig. 6, the concentration of cellobiose from 0 mM to 0.029 mM basically coincides with the abscissa.

The experimental results in Figure 6 show that the proliferation rate of phage is directly proportional to the concentration of cellobiose, and the reduction of cellobiose concentration will slow down its proliferation rate, that is, in the process of PACE evolution, with the continuous decrease of cellobiose concentration, LacY must Positive mutations are generated to improve their cellobiose transport efficiency to package more positive mutant SP-lacY progeny. On the contrary, wild-type SP-lacY and negative mutant SP-lacY are evolved and diluted in rounds. keeps disappearing.

Example 3

Substrate specificity of PACE evolved LacY

1) The host S1030-AP1/AP2/MP was cultured to the logarithmic phase (OD ₆₀₀ =0.4).

2) The first round of evolution, in the 1mL evolution system, the initial final concentration of cellobiose was 29mM, the initial wild-type SP-lacY phage was 1.2×10 ⁵ pfu/mL, the final concentration of arabinose was always 1%, and the host S1030-AP1 was supplemented /AP2/MP to 1mL, 37°C, 150RPM, culture and evolve for 1h. Sampling, serial dilution, mixed 10 μL of diluted phage with 190 μL logarithmic phase host S1030-AP1/AP2/MP, placed at 37°C for 15 minutes, mixed with 1mL of 0.5% soft agar containing 0.5% cellobiose at 50°C, and evenly Spread on a solid agar plate with a diameter of 60 cm, let it stand for 10 minutes, and culture it overnight at 37°C after solidification, calculate the number of phage plaques, and determine the concentration of phage in the system. Take a single plaque, use primers SP1-F and SP1-R to amplify the LacY gene fragment on the phage, and send it to BGI for sequencing to check the mutation status.

3) For the second round of evolution, take 100 μL of the supernatant after centrifugation of the first round of evolution system as the phage SP-lacY to be evolved (the phage at this time is a mixed phage of wild-type SP-lacY and its various mutants), fiber The final concentration of disaccharide was 14.5mM, and the final concentration of arabinose was always 1%. Fresh host S1030-AP1/AP2/MP was added to 1mL, and cultured at 37°C and 150RPM for 1h. Take samples to calculate the phage concentration in the system and detect mutations.

4) By analogy, in the third round, take 100 μL of the supernatant of the second round to continue the evolution, and reduce the concentration of cellobiose, and the fourth round until the last round is the same.

5) In the process of evolution, by calculating the concentration of phages in each round of the system, check the proliferation of phages. If the proliferation is fast, the evolution experiment can be carried out in the next round with 10 μL instead of 100 μL in the previous round; if the proliferation is slow, the Under the concentration of cellobiose, the number of evolution rounds was increased.

6) As the concentration of cellobiose decreased rapidly, the screening pressure became greater, and the evolution time of 1 h was not conducive to the accumulation of mutations. From the 12th round, the evolution time was adjusted to 2 h.

7) A total of 53 rounds of evolution were performed, and the concentration of cellobiose decreased from 29 mM to 100 nM.

8) The evolution results show (Fig. 7) that when the cellobiose concentration is higher than 29 μM, the screening pressure is small, and almost no mutations are generated. Further reducing the cellobiose concentration, many mutation sites appear on LacY, among which the A177V mutation site As the evolution progressed, the points were obviously accumulated, and finally all the mutants contained the mutation of the A177V site, indicating that this site is crucial for the activity of LacY in transporting cellobiose.

Example 4

Verification of the transport activity of the evolution product LacYA177V

1) By comparing the proliferation rate of wild-type SP-lacY and SP-lacYA177V at the same cellobiose concentration, the cellobiose transport activity of the evolved product LacYA177V was verified.

2) The host S1030-AP1/AP2/MP LB medium was cultured to logarithmic phase (OD ₆₀₀ =0.4).

3) Dilute the wild-type SP-lacY and SP-lacYA177V phages to the same concentration, and add them to the above-mentioned logarithmic phase hosts respectively, with the final cellobiose concentration of 290 μM or 58 μM.

4) Cultivate at 37° C. and 150 rpm, take samples every 15 minutes, and detect the phage proliferation in the system.

5) To compare whether the proliferation of wild-type SP-lacY and SP-lacYA177V is different under the same cellobiose concentration.

6) The results showed (Figure 8) that at the same concentration of cellobiose, the proliferation rate of SP-lacYA177V was significantly higher than that of wild-type SP-lacY, indicating that the mutation at site A177V significantly improved the activity of LacY in transporting cellobiose, that is, this set The optimized PACE evolution system can be used to evolve the activity of membrane proteins.

In addition, replacing the lactose transporter with other sugar transporters or with receptor proteins, ion channel proteins, etc., can effectively achieve the purpose of continuous directed evolution.

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Shenzhen Institute of Advanced Technology

<120> A plasmid and phage-assisted continuous directed evolution system and directed evolution method

<130> 2018

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 5579

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aagtaccaca cggaatatac ctaaacttaa ttaacggcac tcctcagcaa atataatgac 3600

cctcttgata acccaagagg gcatttttta atgcccatgg aagggcctcg tgatacgcct 3660

atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 3720

gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 3780

gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 3840

tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3900

tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 3960

gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4020

acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4080

tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4140

gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4200

tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 4260

accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 4320

ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 4380

agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 4440

gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 4500

ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 4560

tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 4620

ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 4680

gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgattaaa 4740

acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4800

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4860

atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4920

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4980

tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 5040

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5100

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gtagttacc 5160

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5220

aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5280

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5340

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5400

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 5460

cagcaacgcg gccgctaggt ctagggcggc ggatttgtcc tactcaggag agcgttcacc 5520

gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttg 5579

<210> 2

<211> 3428

<212>DNA

<213> Artificial sequence

<400> 2

ctttacagct agctcagtcc tagggactgt gctagcgaat tctagagaaa gaggagaaac 60

tcgagatgga acgtcgccgt cgcccgaccc tggaaatggt tgcagccctg gccggtgtct 120

gtcgtggtac ggtgagccgc gttattaacg gtagcgatca ggtctctccg gcgacccgtg 180

aagccgtgaa acgcgcaatc aaagaactgg gctatgtgcc gaatcgtgca gctcgtaccc 240

tggtgacccg tcgtaccgat acggttgcac tggtggtttc tgaaaacaat cagaaactgt 300

ttgctgaacc gttctacgcg ggtattgtgc tgggtgttgg tgtcgcactg agcgaacgtg 360

gctttcaatt cgttctggca accggccgtt ctggtatcga acatgaacgc ctgggcggtt 420

atctggcagg ccagcatgtc gatggtgtgc tgctgctgtc actgcaccgc gatgacccgc 480

tgccgcaaat gctggacgaa gcgggcgttc cgtatgtcta tggcggtcgt ccgctgggtg 540

tgccggaaga acaggtgtcg tacgttgata ttgacaacat cggtggtggc cgtcaggcaa 600

cccaacgtct gattgaaacg ggtcaccgtc gtattgcaac catcgcaggt ccgcaggata 660

tggtcgctgg cgtggaacgt ctgcaaggtt atcgcgaagc cctgctggcg gccggtatgg 720

aatacgacga aaccctggtt agttatggcg attttacgta cgactccggt gtcgcagcta 780

tgcgtgaact gctggatcgt gcgccggatg ttgacgcagt cttcgcagcc agtgacctga 840

tgggcctggc agctctgcgt gttctgcgtg cttccggtcg tcgcgtcccg gaagatgtgg 900

cagtcgtggg ttatgatgac tcaaccgtgg cagaacatgc tgaaccgccg atgacctcgg 960

ttaatcagcc gacggaactg atgggtcgtg aaatggcgcg cctgctggtg gatcgtatca 1020

ccggtgaaac cacggaaccg gtgcgcctgg ttatggaaac gcacctgatg gttcgtgaat 1080

caggctaact gcaggtccct aagtctcctc agcaaaacga aaggcccagt ctttcgactg 1140

agcctttcgt tttatttgac cggatgtcct cttgttcatc atcagtaacc cgtatcgtga 1200

gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg 1260

aggcatcagt gaccaaacag gaaaaaaccg ccttaacat ggcccgcttt atcagaagcc 1320

agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct 1380

gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg 1440

atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 1500

cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 1560

gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc 1620

atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 1680

aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 1740

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 1800

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 1860

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 1920

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 1980

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2040

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 2100

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2160

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2220

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2280

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 2340

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga ggtggtagct cttgatccgg 2400

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2460

aaaaaaagga tctcaaacgg cctatttggc ctatttttct aaatacattc aaatatgtat 2520

ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 2580

agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag 2640

cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc 2700

ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca 2760

acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag 2820

attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat 2880

ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc 2940

ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat 3000

agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat 3060

ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc 3120

gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa 3180

atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag 3240

cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg 3300

cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc 3360

ggcaaataaa cgccatggca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 3420

tttggtac 3428

<210> 3

<211> 14

<212>DNA

<213> Artificial sequence

<400> 3

tgggagcgct ccca 14

Claims (8)

1. A plasmid, characterized in that it comprises an AP1 plasmid and an AP2 plasmid; The AP1 plasmid carries the gIII gene, and a functional protein recognition site is set between the promoter of the plasmid and the ribosome binding site; AP2 plasmid carries functional protein gene; The promoter is the J23109 promoter; The functional protein recognition site in the AP1 is recognized by the functional protein encoded by the functional protein gene of the AP2 plasmid, which plays a repressive effect; the gene sequence of the AP1 plasmid is shown in SEQ ID NO.1, and the AP2 plasmid The gene sequence is shown in SEQID NO.2. 2. the phage-assisted continuous directed evolution system containing the AP1 plasmid and the AP2 plasmid according to claim 1. 3. The phage-assisted continuous directed evolution system according to claim 2, characterized in that, the continuous directed evolution system further comprises a phage, a host bacterium, and a mutagenic plasmid for replacing the gIII gene with a target gene. 4. The phage-assisted continuous directed evolution system according to claim 2, characterized in that the AP1 plasmid and the AP2 plasmid according to claim 1 exist in the form of being transferred into a host bacterium. 5. phage-assisted continuous directed evolution system according to claim 3, is characterized in that, described target gene comprises the gene of membrane protein; The membrane proteins include transport proteins, receptor proteins, and ion channel proteins. 6. The phage-assisted continuous directed evolution system according to any one of claims 3-5, wherein the host bacterium is Escherichia coli carrying F factor; the host bacterium is E.coli S1030. 7. adopt the directed evolution method that the phage-assisted continuous directed evolution system described in any one of claim 3-6 carries out, it is characterized in that, AP1 plasmid, AP2 plasmid and mutagenic plasmid are transferred in described host bacterium, obtain evolutionary host bacteria; The evolutionary host bacteria and the phage whose gIII gene is replaced by the target gene are subjected to multiple rounds of culture and screening under the condition that the screening pressure gradually increases to obtain mutants. 8. The directed evolution method according to claim 7, wherein the target gene is a lactose transporter gene, and the screening pressure is carried out by controlling the concentration of the extracellular substrate cellobiose, and the screening pressure gradually changes Dawei: The concentration of the extracellular substrate cellobiose decreased gradually from 29mM to 29μM.

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