CN109943581A - A plasmid and phage-assisted continuous directed evolution system and directed evolution method - Google Patents

Abstract

The present invention relates to the field of directed evolution of membrane proteins, in particular to a plasmid- and phage-assisted continuous directed evolution system and directed evolution method. 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 promoter of the plasmid and the ribosome binding site; the AP2 plasmid carries the function protein gene. The present invention designs new auxiliary plasmids AP1 and AP2, couples the transport activity of target protein to intracellular and extracellular substrate molecules with the expression of gIII on AP1, and couples the reproductive ability of SP with the activity of the target protein in this indirect way , to achieve the effect of continuous directed evolution of the target protein.

Description

A plasmid and phage-assisted continuous directed evolution system and directed evolution method

technical field

The present invention relates to the field of directed evolution of membrane proteins, 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, this ratio shows the importance of membrane proteins in the organism. Membrane proteins mainly include signal receptors, transport proteins, ion channel proteins and some enzymes, which are essential for 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 of the instability and insolubility of membrane proteins, it is difficult for researchers to obtain high purity The three-dimensional structure of membrane proteins was analyzed and studied. 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.

The traditional methods of mutagenesis and gene 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 liposome display and other methods are complicated to operate, and an expensive in vitro translation system needs to be introduced, which is costly (Biophysics, 2015, 11:67-72).

Harvard University's David R Liu team invented a phage-assisted continuous evolution system (PACE). The system mainly consists of three parts. The first part places the genes that need 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 phages; the second part puts SP The gIII gene required for infection and generation of 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 SP, that is, the reproductive ability of SP in cells is related to the target gene. The biological activities are coupled together; the third part is the mutagenic plasmid MP, which uses the arabinose inducer to induce the expression of the genes DNAQ926, dam and seqA on the MP, so that the wrong bases cannot be repaired during DNA replication, 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 SP will be produced. If the target gene on SP obtains a forward mutation, the expression of the gIII protein on the AP plasmid can package the infective progeny SP phage and secrete it outside the cell, and enter the next round of infection, mutation, progeny Cycle; 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 will not be secreted to the outside of the cell or the number is small; the evolution pool is continuously diluted at a certain flow rate, and positive mutation The SPs of the genotype can continue to produce offspring and remain in the pool, and the SPs with negative mutations are continuously flowed out of the pool and disappear. David R Liu's team used the PACE system to successfully evolve 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 endow these proteins with new properties, but they do not involve the field of membrane proteins, and the existing PACE system is not suitable for directed evolution of membrane proteins.

In view of this, the present invention is proposed.

SUMMARY 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 the gIII protein on AP1, the transporter transports the substrate into the cell, and the substrate molecule can relieve the inhibitory effect of the functional protein, which is about to be transported The transport activity of the protein to extracellular substrate molecules is coupled with the expression of gIII on AP1. In this indirect way, the reproductive capacity of the phage is coupled with the transport activity of the transporter to achieve the effect of continuous directed evolution of the target protein.

In order to realize the above-mentioned purpose of the present invention, the following technical solutions are specially adopted:

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;

AP2 plasmids carry functional protein genes.

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

Further, the promoter is the 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 to play a repressive effect.

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

The functional protein set in the present invention is used to respond to the membrane protein substrate molecules 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, for example, the target gene is a 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 in which the target gene replaces the gIII gene.

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

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

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

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

The host strain is preferably E. coli S1030.

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

The evolution host bacteria and the phage in which the gIII gene is replaced by the target gene are subjected to multiple rounds of culture screening under the condition that the screening pressure gradually increases to obtain a mutant.

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

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

The screening pressure was performed by controlling the concentration of the extracellular substrate cellobiose, and the screening pressure gradually increased: the concentration of the extracellular substrate cellobiose was gradually decreased from 29 mM to below 29 μM.

The step-by-step speed can be reduced by a ratio of 2-15 times, such as 2 times speed, 4 times speed, 6 times speed, 8 times speed, 10 times speed, 12 times speed, 15 times speed and so on.

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

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention designs new auxiliary plasmids AP1 and AP2, which couples the transport activity of the target protein to intracellular and extracellular substrate molecules with the expression of gIII on AP1, and through this indirect method, the reproductive capacity of the phage is related to the target protein's ability to reproduce. Activity coupling to achieve the effect of continuous directed evolution of the target protein.

(2) The phage-assisted continuous directed evolution system provided by the present invention can be effectively used for the continuous directed evolution of the target protein after verification.

(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 transport proteins, receptor proteins, ion channel proteins and the like.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art.

1 is a schematic diagram of the continuous directed evolution of phage-assisted cellobiose by taking lacY as an example provided in 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 bacteriophage SP-lacY in Example 1 of the present invention;

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

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

FIG. 8 is a linear relationship diagram of the transport activity between the mutants obtained in Example 4 of the present invention and the control group.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

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

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

Taking LacY as an example, the schematic diagram of its cellobiose transport activity evolved with 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 promoted by the J23109 promoter. There is a recognition site for the functional protein CelR between the promoter and RBS. In the absence of intracellular cellobiose, the expression of gIII is in a suppressed state. AP2 (SEQ ID NO. 2) carries the CelR protein gene. Both the mutagenic 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. 2014 March; 10(3):216-222).

The host bacteria in the present invention is not limited to E. coli S1030, as long as the E. coli carrying the F factor can be used.

Packing wild-type SP-lacY (SEQ ID NO. 3) The SP carrying lacY was transformed into S1030-AP1/AP2 competent cells, recovered for 2 h and added with 1% cellobiose, and the recovered bacteria were mixed with soft agar and spread evenly on solid agar-containing plates. 37 ℃ overnight, observe the plaque and take a single plaque on the plate to 5mL log phase (OD ₆₀₀ =0.4) S1030-AP1/AP2/MP host bacteria, supplement the final concentration of 1% cellobiose, 37 Incubate at 150rpm for 6h. The culture medium was centrifuged, and the supernatant was filtered with a 0.22 μM filter, which was the wild-type SP-lacY phage, and the titer of the wild-type SP-lacY phage was verified. To test the relationship between cellobiose concentration and phage SP-lacY reproduction rate, only when SP-lacY can respond to the extracellular cellobiose concentration, can the cellobiose concentration be continuously reduced during the evolution of PACE, increase the screening pressure, and make the evolution move towards The direction of increasing the activity of lacY on cellobiose transport was carried out. 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 1 mL, 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 taken as the starting phage and replaced with fresh S1030-AP1/AP2/MP (OD ₆₀₀ = 0.4) to concentrate the mutation on the phage, cellobi 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, and the cellobiose gradually decreased. The evolution time of each round was 1h in the early stage and 2h in the later stage. The phage titer was detected in each round of sampling and sequenced to detect the LacY mutation.

The embodiments of the present invention are described by taking the lactose transporter LacY as an example.

Example 1

Packaging of bacteriophage SP-lacY carrying genes to be evolved

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

2) Construction of the expression plasmid AP2 of the functional protein CelR protein: CelR is expressed from a constitutive promoter (Fig. 3).

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

4) S1030 competent cells were co-transformed with AP1, AP2 and MP to obtain the host S1030-AP1/AP2/MP. Before the host transported cellobiose into the cell without LacY protein, the CelR protein expressed on AP2 and AP1 The CelR recognition site is combined to repress the expression of the downstream gene gIII; MP is a plasmid that enhances the mutation and is induced by arabinose, and its gene sequence is the same as the plasmid IP in the application number 201610349254.3.

5) PCR amplification of the M13 phage macroframe SP (excluding the gIII gene), PCR amplification of the wild-type lacY gene, gibson ligation of the M13 phage macroframe SP and lacY gene to form a SP-lacY double-stranded plasmid, and the ligated product for later use. The gene map of SP-lacY is shown in Figure 5.

6) The ligation product SP-lacY in step 5) transforms the host S1030-AP1/AP2/MP competent cells, recovers for 2 hours and adds cellobiose with a final concentration of 1%. In the recovery stage, LacY transports cellobiose into the cells, and the cells are recovered. Endo-cellobiose relieves the inhibitory effect of CelR protein on gIII protein on AP1, gIII is expressed, the phage is packaged intracellularly and secretes the progeny SP-lacY phage with infection ability, and the recovered bacteria are mixed with soft agar and spread evenly on solid agar plates.

7) Overnight at 37°C, take a single plaque on the plate and put it in 5mL log phase (OD ₆₀₀ = 0.4) S1030-AP1/AP2/MP host bacteria, supplement with a final concentration of 1% cellobiose, 37°C, Incubate at 150rpm for 6h. The culture medium was centrifuged and the supernatant was filtered through a 0.22 μm filter, which was the wild-type SP-lacY phage, and it was verified that the titer of the wild-type SP-lacY phage was 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, restriction enzyme ligation, gene recombination, etc. with reference to the gene map and sequence. These molecular cloning techniques are well known in the art , the corresponding Escherichia coli and phage, templates can be accurately obtained. Therefore, the host bacteria, plasmids, phages, etc. 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 cellobiose concentration and phage SP-lacY reproduction rate

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

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

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

4) Sampling every 15 minutes for each cellobiose gradient to detect the proliferation of phage SP-lacY in the system, and the results are shown in FIG. 6 .

In Fig. 6, the cellobiose concentrations from 0 mM to 0.029 mM are all substantially coincident with the abscissa.

The experimental results in Fig. 6 show that the proliferation rate of phage is proportional to the concentration of cellobiose. When the concentration of cellobiose is reduced, the proliferation rate becomes slower, that is, as the concentration of cellobiose continues to decrease during the evolution of PACE, LacY must A positive mutation was generated to improve the transport efficiency of cellobiose to package more positive mutant SP-lacY progeny. On the contrary, wild-type SP-lacY and negative mutant SP-lacY were evolved and diluted in rounds keeps disappearing.

Example 3

PACE evolves the substrate specificity of LacY

1) Host S1030-AP1/AP2/MP was cultured to log phase (OD ₆₀₀ =0.4).

2) In the first round of evolution, in a 1 mL evolution system, the initial final concentration of cellobiose was 29 mM, 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 added. /AP2/MP to 1mL, 37°C, 150RPM for 1h. Sampling, serially diluted, 10 μL of the diluted phage was mixed with 190 μL log-phase host S1030-AP1/AP2/MP, placed at 37°C for 15 min, and mixed with 1 mL of 0.5% soft agar containing 0.5% cellobiose at 50°C. Spread on a solid agar plate with a diameter of 60 cm, let it stand for 10 min, and then place it at 37 °C for overnight culture after solidification. Calculate the number of 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.

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

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

5) In the process of evolution, by calculating the concentration of phage in each round of the system, check the proliferation of phage. If the proliferation is fast, the next round can take 10 μL of the previous round instead of 100 μL to perform the evolution experiment; if the proliferation is slow, the Round cellobiose concentration increases the number of evolution rounds.

6) With the rapid decrease of cellobiose concentration, the screening pressure increased, and the evolution time of 1h was not conducive to the accumulation of mutations. The 12th round began to adjust the evolution time to 2h.

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

8) The evolution results show (Figure 7) that when the cellobiose concentration is higher than 29 μM, the screening pressure is small, almost no mutation occurs, and the cellobiose concentration is further reduced. Many mutation sites appear on LacY, among which the A177V mutation As evolution progressed, points were significantly accumulated, and finally all mutants contained mutations in the A177V site, indicating that this site is critical for the activity of LacY to transport cellobiose.

Example 4

Validation of the transport activity of the evolution product LacYA177V

1) The cellobiose transport activity of the evolution product LacYA177V was verified by comparing the proliferation rates of wild-type SP-lacY and SP-lacYA177V at the same cellobiose concentration.

2) The host S1030-AP1/AP2/MP LB medium was cultured to log 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 log phase host respectively, and the final concentration of cellobiose is 290 μM or 58 μM.

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

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

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

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

Although specific embodiments of the present invention have been illustrated and described, it should be understood that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications as fall within the scope of this invention be included in the appended claims.

SEQUENCE LISTING

<110> Shenzhen Advanced Technology Research Institute

<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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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 ctttttttgcg 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 ttgatttaaa 4740

acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4800

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4860

atcttcttga gatcctttttt 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 gatagttacc 5160

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5220

aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5280

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5340

gagggagctt ccaggggggaa 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 sequences

<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 ttattttgac cggatgtcct cttgttcatc atcagtaacc cgtatcgtga 1200

gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg 1260

aggcatcagt gaccaaacag gaaaaaaccg cccttaacat 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 sequences

<400> 3

tgggagcgct ccca 14

Claims (10)

1. a plasmid, is characterized in that, comprises one or both in AP1 plasmid and 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 plasmids carry functional protein genes. 2. plasmid according to claim 1, is characterized in that, described 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 to play a repressive effect. 3 . The plasmid according to claim 1 , wherein 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. 4 . 4. A phage-assisted continuous directed evolution system comprising the AP1 plasmid and the AP2 plasmid of any one of claims 1-3. 5 . The phage-assisted continuous directed evolution system according to claim 4 , wherein the continuous directed evolution system further comprises phages, host bacteria and mutagenic plasmids in which the target gene replaces the gIII gene. 6 . 6 . The phage-assisted continuous directed evolution system according to claim 4 , wherein the AP1 plasmid and the AP2 plasmid according to any one of claims 1 to 3 exist in the form of transformed host bacteria. 7 . 7. The phage-assisted continuous directed evolution system according to claim 5, wherein the target gene comprises a membrane protein gene; Preferably, the membrane proteins include transport proteins, receptor proteins, and ion channel proteins. 8. The phage-assisted continuous directed evolution system according to any one of claims 5-7, wherein the host bacteria is Escherichia coli carrying F factor; the host bacteria is preferably E.coli S1030. 9. the directional evolution method that adopts the phage-assisted continuous directional evolution system described in any one of claim 5-8 to carry out, it is characterized in that, AP1 plasmid, AP2 plasmid and mutagenic plasmid are transferred in described host bacteria, obtain Evolutionary host bacteria; The evolution host bacteria and the phage in which the gIII gene is replaced by the target gene are subjected to multiple rounds of culture screening under the condition that the screening pressure gradually increases to obtain a mutant. 10. The directed evolution method according to claim 9, wherein the target gene is a lactose transporter gene, the screening pressure is performed by controlling the concentration of the extracellular substrate cellobiose, and the screening pressure gradually changes. Dawei: The concentration of the extracellular substrate cellobiose gradually decreased from 29mM to below 29μM.