CN109943581B - 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

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 a directed evolution method.

Background

In the genome, approximately 30% of the gene products are membrane proteins, and this ratio indicates the importance of membrane proteins in an organism. The membrane proteins mainly comprise signal receptors, transport proteins, ion channel proteins and enzymes, and are important for cell metabolism, physiological balance and intracellular regulation. In the process of drug research and design, a lot of membrane proteins are target points of drug design, however, the development of drug design is restricted by the shortage of membrane protein structures and biochemical information, and scientific researchers are difficult to obtain high-purity membrane proteins to analyze and research three-dimensional structures of the membrane proteins due to the instability and insolubility of the membrane proteins. Directed evolution or licensing as a powerful tool to address these properties of membrane proteins helps researchers understand the relationships between membrane protein structures and their biological functions.

The conventional methods of mutagenesis and gene recombination are time-consuming and labor-consuming, and require the construction of mutation libraries and repeated screening (Journal of clinical Biology,2000,205 (3): 483-503). Current newer liposome displays are complicated to operate, require the introduction of expensive in vitro translation systems, and are costly (Biophysics, 2015, 11.

The David R Liu team at Harvard university invented a phage assisted continuous evolution system (PACE). The system mainly comprises three parts, wherein in the first part, a gene to be evolved is placed on a bacteriophage M13 genome and replaces an original gIII gene to form SP, and the SP lacks gIII and cannot infect a host and generate a progeny bacteriophage; the second part puts gIII gene needed by SP infection and generation of progeny phage on extra plasmid to express, forming assistant plasmid AP, the expression of gIII is controlled by the activity of objective gene on SP, that is, the reproductive capacity of SP in cell is coupled with the biological activity of objective gene; the third part is mutagenic plasmid MP, and arabinose inducer is used to induce the expression of genes DNAQ926, dam and seqA on MP, so that the wrong base can not be repaired in the process of DNA replication, and the mutation rate is increased by hundreds of times. When the SP carrying the gene of interest infects the host, the SP replicates the genetic material in the host cell and produces various mutants of SP with the help of MP. If the objective gene on SP obtains positive mutation, gIII protein on AP plasmid is expressed, and filial generation SP bacteriophage with infectivity can be packaged and secreted to the extracellular space, and then enter the next infection, mutation and filial generation circulation; if the objective gene on SP is not mutated or is negatively mutated, the gIII protein on the AP plasmid is not expressed, and the generated filial generation is not secreted to the extracellular or is small in quantity; the evolution pool is diluted continuously at a certain flow rate, the SP with positive mutation can continuously generate filial generation and is retained in the pool, and the SP with negative mutation can continuously flow out of the pool and disappear. The group of David R Liu successfully evolved T7RNA polymerase (NATURE.2011April; 472-498-505), protease (Nature Communications,2014,5, 5352.), DNA binding protein (Nature Methods,2015,12 (10): 939.), etc. and impart new properties to these proteins, but do not relate to the field of membrane proteins, and the existing PACE systems are not suitable for directed evolution of membrane proteins.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention designs new auxiliary plasmids AP1 and AP2, wherein the protein expressed by the functional gene on the AP2 inhibits the expression of gIII protein on the AP1, the transporter transports a substrate into cells, and the substrate molecule can relieve the inhibition effect of the functional protein, namely the transport activity of the transporter on extracellular substrate molecules is coupled with the gIII expression on the AP1, and the reproductive capacity of the bacteriophage is coupled with the transport activity of the transporter in an indirect mode, so that the effect of continuously and directionally evolving the target protein is achieved.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a plasmid, comprising one or two of an AP1 plasmid and an AP2 plasmid;

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

the AP2 plasmid carries a functional protein gene.

The AP1 plasmid provided by the invention is an auxiliary plasmid expressed by a phage infection and multiplication essential gene gIII; the AP1 plasmid carries the gIII gene and is driven by a promoter such as the J23109 promoter, which has a recognition site for functional proteins between the promoter and the RBS, and gIII expression is repressed in the absence of an inducer intracellularly. The AP2 plasmid is a helper plasmid that indirectly couples the proliferative capacity of the phage to the concentration of extracellular substrate molecules and the activity of membrane proteins such as transporters.

Further, the promoter is a J23109 promoter.

Preferably, the functional protein recognition site in the AP1 is recognized by a functional protein encoded by a functional protein gene of the AP2 plasmid, serving as a repression effect.

That is, 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 achieving a repression effect.

The functional protein provided by the invention is used for responding to membrane protein substrate molecules transported into cells, relieving the repression effect and inducing the expression of the gIII gene.

In the invention, different functional proteins correspond to different substrate molecules, and the different substrate molecules are used for evolving different target genes, for example, the target gene is a lactose transporter gene, the substrate molecule is disaccharide, and the functional protein is functional protein CelR.

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

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

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

Furthermore, the continuous directed evolution system also comprises a phage, a host bacterium and a mutagenic plasmid, wherein the gIII gene is replaced by the target gene.

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

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

preferably, the membrane protein comprises a transporter protein, a receptor protein, an ion channel protein.

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

Coli S1030.

The invention also provides an directed evolution method which is carried out by adopting the phage-assisted continuous directed evolution system, and AP1 plasmid, AP2 plasmid and mutagenic plasmid are transferred into the host bacteria to obtain evolved host bacteria;

and carrying out multiple rounds of culture screening on the evolved host bacteria and the phage with the gIII gene replaced by the target gene under the condition that the screening pressure is gradually increased to obtain the mutant.

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

Further, the gene of interest is a lactose transporter gene, and the

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

The step-down speed may be: the reduction is in a proportion 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, etc.

In the method, 1/10 volume of supernatant in each round of multi-round culture can be taken as a starting phage in the next round, and fresh evolved host bacteria are replaced in each round to ensure that mutation is accumulated on a target gene on the phage; the proliferation of the phage is induced by the addition of substrate molecules for membrane proteins or transporters per round of culture. The multi-round culture screening process of the invention is to improve the screening pressure by continuously reducing the molecular concentration of substrates such as membrane protein or transport protein, so that the evolution direction is oriented to improve the transport efficiency of target genes to substrate molecules, thereby realizing the evolution of target genes.

Compared with the prior art, the invention has the beneficial effects that:

(1) 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 phage with the activity of target protein in an indirect mode, and achieves the effect of continuously and directionally evolving target protein.

(2) The phage-assisted continuous directed evolution system provided by the invention can be effectively used for continuous directed evolution of target proteins by experiments.

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

Drawings

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

FIG. 1 is a schematic diagram of phage-assisted directed evolution of cellobiose, using lacY as an example, according to an embodiment of the present invention;

FIG. 2 is a schematic genetic map of helper plasmid AP1 in example 1 of the present invention;

FIG. 3 is a gene map of helper plasmid AP2 in example 1 of the present invention;

FIG. 4 is a schematic diagram of the genetic map of the mutagenized plasmid MP in example 1 of the present invention;

FIG. 5 is a schematic genetic map of phage SP-lacY in example 1 of the present invention;

FIG. 6 is a graph showing the linear relationship between the proliferation rate of phage and the concentration of extracellular cellobiose in example 2 of the present invention;

FIG. 7 is a schematic diagram of the identification of the LacY mutation site by phage-assisted continuous directed evolution (CFE) system in example 3 of the present invention;

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

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The currently designed PACE systems are not available for directed evolution of membrane proteins, particularly transport proteins. The phage-assisted continuous directed evolution system provided by the invention comprises a phage SP carrying a pseudo-evolution transporter gene, an arabinose-induced mutant plasmid MP, an assistant plasmid AP1 expressed by an SP infection and multiplication essential gene gIII, and an assistant plasmid AP2 for regulating and controlling infection resistance of a host phage and indirectly coupling SP multiplication capacity with extracellular substrate molecule concentration and transporter activity. The invention can be used for the directed evolution of various membrane proteins and transport proteins.

The lactose transporter LacY is taken as an example for explanation, and the transport activity of LacY on cellobiose is improved through PACE evolution, but the protection of the invention is not limited to the transporter LacY and the evolution of the transport activity of the transporter LacY on cellobiose, such as the evolution of LacY on other saccharide transport activities, the evolution of the transport activity of other transporters or ion channel proteins on substrates thereof, and the like.

A schematic diagram of the transport activity of LacY, which was evolved using the modified PACE system, for cellobiose is shown in FIG. 1.

SP carries the gene of interest lacY to be evolved. AP1 (SEQ ID NO. 1) carries gIII gene and is started by J23109 promoter, a recognition site of functional protein CelR is arranged between the promoter and RBS, and gIII expression is in a suppression state under the condition that cellobiose is not in cells. AP2 (SEQ ID NO. 2) carries the CelR protein gene. Both the mutagenized plasmid MP and the host strain S1030 are provided by David R Liu laboratories, and their genetic information has been reported in the relevant literature (Nat Chem biol.2014March;10 (3): 216-222).

Coli S1030, any escherichia coli carrying factor F may be used.

Packaging wild SP-lacY (SEQ ID NO. 3), transforming S1030-AP1/AP2 competent cells with SP carrying lacY, recovering for 2h, adding 1% cellobiose, mixing the recovered bacteria with soft agar, and spreading on a plate containing solid agar. Overnight at 37 ℃, plaques were observed and individual plaques on the plate were removed in a 5mL log phase (OD) ₆₀₀ = 0.4) S1030-AP1/AP2/MP host cells, supplemented with cellobiose at a final concentration of 1%, cultured at 37 ℃ for 6h at 150 rpm. Centrifuging the culture solution, and filtering the supernatant with 0.22 μ M filter membraneThe wild-type SP-lacY phage titer was verified for the wild-type SP-lacY phage. The relationship between cellobiose concentration and bacteriophage SP-lacY propagation speed is tested, and only when the SP-lacY can respond to extracellular cellobiose concentration, the cellobiose concentration can be continuously reduced in the PACE evolution process, the screening pressure is improved, and the evolution is carried out towards the direction of improving the transport activity of lacY on cellobiose. The LacY evolved by PACE, the initial phage was wild-type SP-lacY with a titer of 1X 10 ⁵ pfu/mL, host S1030-AP1/AP2/MP (OD) ₆₀₀ = 0.4), 1mL of evolution system, 1% arabinose was always maintained, and the first round cellobiose concentration was 1%; the second round was performed by replacing fresh S1030-AP1/AP2/MP (OD) with 100. Mu.l of supernatant from the first round as starting phage ₆₀₀ = 0.4) the mutation was concentrated on the phage, the cellobiose concentration was reduced to 0.5%, and so on, with 10-fold dilution in each round, with fresh host replaced in each round, the cellobiose was gradually reduced, with 1h at the early stage and 2h at the later stage of each round of evolution. Each round of sampling was performed to detect phage titer and sequencing was performed to detect LacY mutations.

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

Example 1

Packaging of phage SP-lacY carrying pseudoevolved genes

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

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

3) The mutagenized plasmid MP was submitted to the David R Liu laboratory and the map is shown in FIG. 4.

4) AP1, AP2 and MP are co-transformed into S1030 competent cells to obtain a host S1030-AP1/AP2/MP, wherein the CelR protein expressed on the AP2 is combined with the CelR recognition site on the AP1 to repress the expression of a downstream gene gIII before the host transfers cellobiose into cells without LacY protein; MP is a mutation-increasing plasmid, induced by arabinose, and the gene sequence is the same as that of plasmid IP in application No. 201610349254.3.

5) PCR amplifies M13 phage large frame SP (not containing gIII gene), PCR amplifies wild type lacY gene, gibson connects M13 phage large frame SP and lacY gene to form SP-lacY double chain plasmid, and the connection product is ready for use. The genetic map of SP-lacY is shown in FIG. 5.

6) And 5) transforming a host S1030-AP1/AP2/MP competent cell by using the connection product SP-lacY, recovering for 2 hours, adding cellobiose with the final concentration of 1%, transferring the cellobiose into cells by using LacY in the recovery stage, removing the inhibition effect of CelR protein on gIII protein on AP1 by using the intracellular cellobiose, expressing the gIII, packaging the phage in the cells, secreting progeny SP-lacY phage with infection capacity, and uniformly mixing the recovered bacteria and soft agar and flatly paving the mixture on a plate containing solid agar.

7) Overnight at 37 ℃ and single plaques on the plate were taken at 5mL log phase (OD) ₆₀₀ = 0.4) S1030-AP1/AP2/MP host cells, supplemented with cellobiose at a final concentration of 1%, cultured at 37 ℃ for 6h at 150 rpm. Centrifuging the supernatant of the culture solution, filtering with 0.22 μm filter membrane to obtain wild SP-lacY phage, and verifying that titer of wild SP-lacY phage is 1 × 10 ¹¹ pfu/mL。

In the invention, the plasmids AP1, AP2, MP and double-chain SP-lacY can be obtained by conventional molecular cloning methods such as PCR, enzyme digestion connection, gene recombination and the like according to a gene map and a sequence, the molecular cloning technologies are well known in the field, and the corresponding escherichia coli, phage and template can be obtained exactly. Therefore, the host bacteria, plasmids, phages and the like of the present invention have reproducible characteristics and can be obtained by those skilled in the art by conventional methods.

Example 2

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

1) The host S1030-AP1/AP2/MP LB medium is cultured to logarithmic phase (0D) ₆₀₀ ＝0.4)。

2) Wild-type SP-lacY phage was diluted and added to the log phase host described above to give an initial concentration of 50pfu/mL in the system.

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

4) The phage SP-lacY proliferation in the system was examined by sampling every 15min for each cellobiose gradient, and the results are shown in FIG. 6.

In FIG. 6, cellobiose at concentrations of 0mM to 0.029mM all substantially coincide with the abscissa.

FIG. 6 shows that the proliferation rate of phage is proportional to the concentration of cellobiose, and the proliferation rate of the phage becomes slower as the concentration of cellobiose is reduced, i.e. during the evolution of PACE, lacY must generate positive mutation to improve the transport efficiency of LacY to cellobiose to pack more filial generation of the positive mutation SP-lacY, whereas wild-type SP-lacY and negative mutation SP-lacY disappear continuously during one round of evolution and dilution.

Example 3

Substrate specificity of PACE evolved LacY

1) The host S1030-AP1/AP2/MP was cultured to log phase (0D) ₆₀₀ ＝0.4)。

2) First round of evolution, 1mL evolution System, initial final cellobiose concentration of 29mM, initial wild type SP-lacY phage 1.2X 10 ⁵ pfu/mL, arabinose final concentration was always 1%, the host S1030-AP1/AP2/MP was supplemented to 1mL, the culture was evolved at 37 ℃ for 1h at 150 RPM. Sampling, diluting in a gradient manner, mixing 10 mu L of diluted phage with 190 mu L of log phase host S1030-AP1/AP2/MP, standing at 37 ℃ for 15min, uniformly mixing with 1mL of 0.5% soft agar containing 0.5% cellobiose at 50 ℃, uniformly paving on a solid agar plate with the diameter of 60cm, standing for 10min, standing at 37 ℃ for overnight culture after solidification, calculating the number of plaques, and determining the concentration of phage in the system. Taking a single plaque, amplifying a LacY gene fragment on the phage by using primers SP1-F and SP1-R, sending to Huada sequencing, and checking the mutation condition.

3) And in the second evolution, taking 100 mu L of supernatant obtained after centrifugation of the first evolution system as phage SP-lacY to be evolved (the phage is wild SP-lacY and mixed phage of various mutants thereof), wherein the final cellobiose concentration is 14.5mM, the final arabinose concentration is always 1%, and the fresh host S1030-AP1/AP2/MP is supplemented to 1mL,37 ℃ and 150RPM for culture and evolution for 1h. Sampling and calculating the concentration of the phage in the system and detecting the mutation condition.

4) By analogy, the third round takes 100 μ L of the supernatant of the second round to continue the evolution, and reduces the cellobiose concentration, and the fourth round is the same until the last round.

5) During the evolution process, the concentration of the phage in each round of the system is calculated, the multiplication condition of the phage is checked, if the multiplication is faster, the next round can take 10 μ L to replace 100 μ L to execute the evolution experiment; if proliferation is slower, the number of evolution rounds is increased at this cellobiose concentration.

6) With the rapid decrease of the cellobiose concentration, the screening pressure is increased, the evolution time of 1h is not beneficial to the accumulation of the mutation, and the evolution time is adjusted to 2h from the 12 th round.

7) Evolution was performed for 53 cycles with cellobiose concentrations decreasing from 29mM to 100nM.

8) The evolution results (fig. 7) show that at cellobiose concentrations above 29 μ M, the screening pressure is low, almost no mutation is generated, the cellobiose concentration is further reduced, a plurality of mutation sites appear on LacY, wherein the a177V mutation site is obviously accumulated as the evolution progresses, and finally all mutants contain the mutation of the a177V site, which indicates that the site is important for the cellobiose transport activity of LacY.

Example 4

Verification of transport activity of evolution product LacYA177V

1) The cellobiose transport activity of the evolutionary product LacYA177V is verified by comparing the proliferation rate of wild SP-lacY and SP-lacYA177V at the same cellobiose concentration.

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

3) Wild-type SP-lacY and SP-lacYA177V phages were diluted to the same concentration and added to the log-phase host at a final cellobiose concentration of 290. Mu.M or 58. Mu.M, respectively.

4) Culturing at 37 deg.C and 150rpm, sampling every 15min, and detecting bacteriophage proliferation condition in the system.

5) Comparing whether the wild SP-lacY and SP-lacYA177V proliferate differently under the same cellobiose concentration.

6) The result shows (figure 8) that under the same cellobiose concentration, the proliferation speed of SP-lacYA177V is obviously higher than that of wild-type SP-lacY, which indicates that the activity of LacY for transporting cellobiose is obviously improved by mutation of site A177V, namely the activity of the optimized PACE evolution system can be used for evolving membrane protein.

In addition, the lactose transporter is replaced by transporters of other saccharides or replaced by receptor proteins, ion channel proteins and the like, so that the aim of continuous directed evolution can be effectively fulfilled.

While particular embodiments of the present invention have been illustrated and described, it would be obvious 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 advanced technology research institute

<120> 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

<212> DNA

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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 ttgatttaaa 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 gatagttacc 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 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 sequence

<400> 3

tgggagcgct ccca 14

Claims (8)

1. A plasmid comprising 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 promoter is a J23109 promoter;

the functional protein recognition site in the AP1 is recognized by the functional protein coded by the functional protein gene of the AP2 plasmid, so that a repression effect is achieved; the gene sequence of the AP1 plasmid is shown as SEQ ID No.1, and the gene sequence of the AP2 plasmid is shown as SEQ ID No. 2.

2. A phage-assisted continuous directed evolution system comprising the AP1 plasmid and the AP2 plasmid of claim 1.

3. The phage-assisted continuous directed evolution system of claim 2, further comprising a phage, a host bacterium, a mutagenized plasmid, wherein the gene of interest replaces the gIII gene.

4. A phage-assisted continuous directed evolution system according to claim 2, wherein the AP1 plasmid and the AP2 plasmid of claim 1 are present in the form of transformed host bacteria.

5. A phage-assisted continuous directed evolution system according to claim 3, wherein the gene of interest comprises a gene of a membrane protein;

the membrane protein comprises transport protein, receptor protein and ion channel protein.

6. A phage-assisted continuous directed evolution system according to any one of claims 3 to 5, wherein the host bacterium is E.coli carrying factor F; the host bacterium is E.coli S1030.

7. Directed evolution process using a phage-assisted continuous directed evolution system according to any of claims 3 to 6, characterized in that the AP1 plasmid, the AP2 plasmid and the mutagenized plasmid are transferred into the host bacterium to obtain an evolved host bacterium;

and carrying out multiple rounds of culture screening on the evolved host bacteria and the phage with the gIII gene replaced by the target gene under the condition that the screening pressure is gradually increased to obtain the mutant.

8. The directed evolution method according to claim 7, wherein the gene of interest is a lactose transporter gene, the selection pressure is performed by controlling the concentration of extracellular substrate cellobiose, and the selection pressure is gradually increased to: the extracellular substrate cellobiose concentration was gradually decreased from 29mM to 29. Mu.M.

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