CN109943581A - The continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary - Google Patents

Abstract

The present invention relates to memebrane protein directed evolution field, in particular to the continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary.A kind of plasmid, including one or both of AP1 plasmid and AP2 plasmid；AP1 plasmid carries gIII gene, and functional protein recognition site is provided between the promoter and ribosome bind site of the plasmid；AP2 plasmid carrying function protein gene.The present invention designs new helper plasmid AP1 and AP2, target protein is expressed into coupling to the gIII on the transport activity and AP1 of outer substrate molecule intracellular, the activity of the fertility of SP and target protein is coupled by this indirect mode, achievees the effect that continuously-directional evolution destination protein.

Description

The continuously-directional evolutionary system and directed evolution of a kind of plasmid and bacteriophage auxiliary Method

Technical field

The present invention relates to memebrane protein directed evolution field, the company assisted in particular to a kind of plasmid and bacteriophage Continuous directed evolution system and directed evolution method.

Background technique

In genome, about 30% gene product is memebrane protein, this ratio shows memebrane protein in organism Importance.Memebrane protein mainly includes frizzled receptor, transport protein, ionophorous protein and some enzymes, to cell metabolism, life Pat weighing apparatus, it is intracellular adjust it is most important.In medicament research and development design process, many memebrane proteins are the target spots of drug design, however The scarcity of Membrane protein conformation and Biochemical Information constrains the development of drug design, because the unstability of memebrane protein and insoluble leading The memebrane protein that cause scientific research personnel is difficult acquisition high-purity carries out the parsing and research of its three-dimensional structure.For these spies of memebrane protein Property, directed evolution perhaps can be used as the pass that strong tool helps scientific research personnel to understand Membrane protein conformation and its biological function System.

The mode of traditional mutagenesis and genetic recombination memebrane protein of evolving takes time and effort, and needs to build mutation library, repeated screening into Row (Journal of Theoretical Biology, 2000,205 (3): 483-503).Current relatively new lipid body display Etc. modes it is complicated for operation, also need to introduce expensive external translating system, higher cost (Biophysics, 2015,11:67-72).

A kind of bacteriophage auxiliary constant evolution system (PACE) has been invented by David R Liu team, Harvard University.The system It mainly include three parts, first part will need the gene evolved to be placed on bacteriophage M13 genome and replace original GIII gene forms SP, and SP, which lacks gIII, can not infect host and generate progeny phage；SP is infected and is generated by second part GIII gene needed for progeny phage is placed on additional plasmid and is expressed, and forms helper plasmid AP, the expression of gIII by The bioactivity of SP fertility in the cell and target gene is coupled at one by the active regulation of target gene on SP It rises；Part III is Mutagenic plasmid MP, utilizes gene DNA Q926, dam and seqA table on arabinose inducer induction MP It reaches, for false bases from repairing, mutation rate improves hundred times during preventing DNA replication dna.When the SP with target gene infects place After master, SP can generate the various mutant of SP with the help of MP in host's duplication intracellular for carrying out inhereditary material.If on SP Target gene obtain forward mutation assay, then the gIII protein expression on AP plasmid, can pack the filial generation SP for providing infectivity Bacteriophage is simultaneously secreted into extracellular, into the infecting of next round, is mutated, filial generation circulation；If the target gene on SP be not mutated or Negative mutation, then the gIII albumen on AP plasmid is not expressed, and the filial generation of generation is not secreted into extracellular or negligible amounts；Evolution pond It is persistently to be diluted with certain flow rate, the SP of front mutation can continue to generate filial generation and be retained in pond, the SP being negatively mutated It can constantly outflow in pond and disappear.David R Liu team has successfully been evolved t7 rna polymerase with PACE system (NATURE.2011April；472:498-505), protease (Nature Communications, 2014,5:5352.), DNA (2015,12 (10): Nature Methods 939.) etc. and assigns these albumen new characteristic, but is not directed to binding protein Memebrane protein field, existing PACE system are not appropriate for being oriented memebrane protein evolution.

In view of this, the present invention is specifically proposed.

Summary of the invention

The present invention designs new helper plasmid AP1 and AP2, and the albumen of the functional gene expression on AP2 inhibits gIII on AP1 The expression of albumen, by substrate transport into intracellular, substrate molecule can will be turned transport protein with the inhibitory effect of removing function albumen It transports albumen and coupling is expressed to the gIII on the transport activity and AP1 of extracellular substrate molecule, by this indirect mode by phagocytosis The fertility of body and the transport activity of transport protein are coupled, and achieve the effect that continuously-directional evolution destination protein.

In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:

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

AP1 plasmid carries gIII gene, and functional egg is arranged between the plasmid promoter and ribosome bind site White recognition site；

AP2 plasmid carrying function protein gene.

AP1 plasmid provided by the invention is the helper plasmid of Phage Infection and proliferation indispensable gene gIII expression；AP1 Plasmid carries gIII gene and is started by promoter such as J23109 promoter, the identification of functional albumen between promoter and RBS Site, it is intracellular do not have inducer in the case where, gIII expression be in holddown.AP2 plasmid is by bacteriophage multiplication ability With the helper plasmid of extracellular substrate molecule concentration and memebrane protein such as transport protein activity indirect conjugation.

Further, the promoter is J23109 promoter.

Preferably, the function that the functional protein recognition site in the AP1 is encoded by the functional protein gene of the AP2 plasmid Energy albumen identification, plays reptation behavior.

That is, the functional protein that the functional protein gene of AP2 plasmid encodes is used for and the functional protein in AP1 plasmid Recognition site combines, and then realizes reptation behavior.

The functional protein being arranged in the present invention is transported for responding into memebrane protein substrate molecule intracellular, derepression effect It answers, induces the expression of gIII gene.

In the present invention, different functional proteins corresponds to different substrate molecules, and different substrate molecules is for difference of evolving Target gene, if target gene be lactose transporter gene, substrate molecule is disaccharides, and functional protein is functional protein CelR。

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

Further, the gene order of the AP1 plasmid is as shown in SEQ ID NO.1, the gene order of the AP2 plasmid As shown in SEQ ID NO.2.

The present invention also provides a kind of continuously-directional evolutionary systems of bacteriophage auxiliary, which contains above-mentioned AP1 plasmid and AP2 plasmid.

Further, the continuously-directional evolutionary system further includes the bacteriophage of target gene replacement gIII gene, host Bacterium, Mutagenic plasmid.

Further, the AP1 plasmid and AP2 plasmid exist in the form of being transferred to host strain.

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

Preferably, the memebrane protein includes transport protein, receptor protein, ionophorous protein.

Further, the host strain is the Escherichia coli for carrying the F factor.

The host strain is preferably E.coli S1030.

The present invention also provides the directed evolution sides that the continuously-directional evolutionary system assisted using above-mentioned bacteriophage is carried out AP1 plasmid, AP2 plasmid and Mutagenic plasmid are transferred in the host strain, obtain evolution host strain by method；

What the bacteriophage of the evolution host strain and target gene replacement gIII gene gradually became larger in screening pressure In the case of carry out more wheel culture screenings, obtain mutant.

The evolvement method provided by the invention, realizes the directed evolution of memebrane protein, for memebrane protein evolution provide it is important Technical support.

Further, the target gene is lactose transporter gene, described

Screening pressure is carried out by controlling the concentration of extracellular substrate cellobiose, and the screening pressure gradually becomes larger are as follows: born of the same parents Outer bottom fibres disaccharides concentration is gradually down to 29 μM or less from 29mM.

The speed gradually reduced can be with are as follows: it is reduced with 2-15 times of ratio, it such as can be with 2 speeds, 4 speeds, 6 speeds Degree, 8 speeds, 10 speeds, 12 speeds, 15 speeds etc..

In this method, every wheels in more wheel cultures can use the supernatant of last round of 1/10 volume and set out phagocytosis as next round Body, each round replace fresh evolution host strain, guarantee mutation accumulation on the target gene on bacteriophage；Every wheel culture passes through Add the proliferation of the substrate molecule inducible phage of memebrane protein or transport protein.More wheel culture screening processes of the invention are to pass through The molecular concentration for constantly reducing substrate such as memebrane protein or transport protein improves screening pressure, orients Evolutionary direction to improve purpose For the purpose of the transfer efficiency of gene pairs substrate molecule, the evolution of target gene is realized.

Compared with prior art, the invention has the benefit that

(1) present invention designs new helper plasmid AP1 and AP2, and transhipment of the target protein to outer substrate molecule intracellular is living Property express and be coupled with the gIII on AP1, by this indirect mode by the activity of the fertility of bacteriophage and target protein Coupling, achievees the effect that continuously-directional evolution destination protein.

(2) the continuously-directional evolutionary system of bacteriophage auxiliary provided by the invention, it is verified, purpose can be effectively used for The lasting directed evolution of albumen.

(3) the continuously-directional evolutionary system of bacteriophage auxiliary provided by the invention, can be used for a variety of memebrane proteins and such as transports egg The directed evolution of white, receptor protein, ionophorous protein etc..

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 be it is provided in an embodiment of the present invention by taking lacY as an example, bacteriophage auxiliary cellobiose continuously-directional evolve Schematic diagram；

Fig. 2 is the genome schematic diagram of helper plasmid AP1 in the embodiment of the present invention 1；

Fig. 3 is the genome schematic diagram of helper plasmid AP2 in the embodiment of the present invention 1；

Fig. 4 is the genome schematic diagram of Mutagenic plasmid MP in the embodiment of the present invention 1；

Fig. 5 is the genome schematic diagram of 1 pnagus medius SP-lacY of the embodiment of the present invention；

Fig. 6 is the linear relationship chart of 2 pnagus medius growth rate and extracellular cellobiose concentration of the embodiment of the present invention；

Fig. 7 is the continuously-directional evolutionary system mutational site the evolution LacY identification of 3 pnagus medius of embodiment of the present invention auxiliary Schematic diagram；

Fig. 8 is the linear relationship chart of the transport activity obtained in the embodiment of the present invention 4 between mutant and control group.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.

The existing PACE system designed can not be used to directed evolution memebrane protein, especially transport protein.The present invention The continuously-directional evolutionary system of the bacteriophage auxiliary of offer, the bacteriophage SP comprising carrying quasi- evolution transporter gene, I The mutant plasmid MP, SP of uncle's sugar induction infect and are proliferated the helper plasmid AP1 of indispensable gene gIII expression, modulate host bacteriophage Infection resistance and by the helper plasmid of SP proliferative capacity and extracellular substrate molecule concentration and transport protein activity indirect conjugation AP2.The present invention can be used for the directed evolution of a variety of memebrane proteins, transport protein.

The present invention is illustrated by taking lactose transport protein LacY as an example, is evolved by PACE and is improved LacY to cellobiose Transport activity, but protection of the invention is not limited to transport protein LacY and its evolution to cellobiose transport activity, such as Evolution of the LacY to other carbohydrate transport activities, other transport proteins or ionophorous protein evolve to the transport activity of its substrate Deng.

By taking LacY as an example, with its transport activity schematic diagram such as Fig. 1 institute to cellobiose of the PACE phyletic evolution after improvement Show.

SP carries the quasi- target gene lacY to evolve.AP1 (SEQ ID NO.1) carries gIII gene and is started by J23109 Son starting, the recognition site of functional PROTEIN C elR between promoter and RBS, it is intracellular do not have cellobiose in the case where, GIII expression is in holddown.AP2 (SEQ ID NO.2) carries CelR protein gene.Mutagenic plasmid MP and host strain S1030 It is provided by the laboratory R Liu David, existing pertinent literature reports its hereditary information (Nat Chem Biol.2014March； 10(3):216–222)。

Host strain in the present invention is not limited to E.coli S1030, as long as the Escherichia coli for carrying the F factor all may be used.

Packaging wild type SP-lacY (SEQ ID NO.3) is thin by the SP conversion S1030-AP1/AP2 competence for carrying lacY The cellobiose of born of the same parents, recovery 2h and addition 1%, the bacterium after recovery is uniformly mixed with soft agar is laid in the plate containing solid agar On.37 DEG C overnight, and single plaque is in 5mL logarithmic phase (OD on observation plaque and plate of making even₆₀₀=0.4) S1030-AP1/ In AP2/MP host strain, the cellobiose of final concentration 1% is added, 37 DEG C, 150rpm cultivates 6h.Medium centrifugal, 0.22 μ of supernatant M membrane filtration, as wild type SP-lacY bacteriophage verify wild type SP-lacY phage titre.It is dense to test cellobiose The relationship of degree and bacteriophage SP-lacY reproduction speed, when only SP-lacY is able to respond extracellular cellobiose concentration, Cai Neng Cellobiose concentration is constantly reduced in PACE evolutionary process, improves screening pressure, makes to evolve towards raising lacY to cellobiose The direction of transport activity carries out.PACE evolution LacY, initial bacteriophage are wild type SP-lacY, titre 1 × 10⁵Pfu/mL, place Main is S1030-AP1/AP2/MP (OD₆₀₀=0.4), evolution system 1mL, arabinose maintain 1% always, first round fiber two Sugared concentration is 1%；Second wheel takes 100 μ l supernatant of the first round as the bacteriophage that sets out, and replaces fresh S1030-AP1/AP2/MP (OD₆₀₀=0.4) mutation being concentrated on bacteriophage, cellobiose concentration is reduced to 0.5%, and so on, each round dilution 10 Times, every wheel replaces fresh host, and cellobiose gradually decreases, and every wheel evolves early period time as 1h, later period 2h.Every wheel sampling inspection It surveys phage titre and detection LacY catastrophe is sequenced.

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

Embodiment 1

Carry the packaging of the bacteriophage SP-lacY of quasi- evolution gene

1) building of gIII protein expressing plasmid AP1: gIII albumen is started by bacteriophage shock promoters J23109, starting The recognition site nucleic acid sequence (SEQ ID NO.3) of sub- downstream insertion CelR albumen.AP1 map is shown in Fig. 2.

2) the expression plasmid AP2 building of functional protein CelR albumen: CelR expresses (Fig. 3) by constitutive promoter.

3) Mutagenic plasmid MP is given by the laboratory David R Liu, and map is shown in Fig. 4.

4) AP1, AP2 and MP cotransformation S1030 competent cell, obtains host S1030-AP1/AP2/MP, which exists There is no LacY albumen to transport cellobiose into the CelR recognition site knot on the CelR albumen and AP1 before intracellular, expressed on AP2 It closes, checks the expression of downstream gene gIII；MP is the plasmid for improving mutation, is induced by arabinose, gene order and application Number for the plasmid IP in 201610349254.3 it is identical.

5) not PCR amplification M13 bacteriophage big frame SP (not including gIII gene), PCR amplification wild type lacY gene, Gibson connection M13 bacteriophage big frame SP and lacY gene forms SP-lacY Double stranded plasmids, and connection product is spare.SP-lacY Genome it is as shown in Figure 5.

6) the 5 of step) connection product SP-lacY conversion host S1030-AP1/AP2/MP competent cell, recovery 2h is simultaneously The cellobiose of final concentration 1% is added, revival phase LacY transports cellobiose into intracellular, cellobiose releasing CelR intracellular Albumen expresses the inhibitory effect of gIII albumen on AP1, gIII, and bacteriophage is in packaging intracellular and secretes with infection ability Filial generation SP-lacY bacteriophage, the bacterium after recovery is uniformly mixed with soft agar to be laid on the plate containing solid agar.

7) it stays overnight for 37 DEG C, single plaque is in the logarithmic phase (OD of 5mL on plate of making even₆₀₀=0.4) S1030-AP1/AP2/MP In host strain, the cellobiose of final concentration 1% is added, 37 DEG C, 150rpm cultivates 6h.0.22 μm of filter membrane mistake of medium centrifugal supernatant Filter, as wild type SP-lacY bacteriophage, verifying wild type SP-lacY phage titre are 1 × 10¹¹pfu/mL。

In the present invention, plasmid AP1, AP2, MP and double-strand SP-lacY bacterium can refer to genome and sequence, pass through routine The molecular cloning methods such as PCR, digestion connection, genetic recombination obtain, these molecule clone technologies are the known of this field, accordingly Escherichia coli and bacteriophage, template can be obtained definitely.Therefore, host strain of the invention, plasmid and bacteriophage etc. have can The feature of repeatability, as long as those skilled in the art conventionally can be obtained.

Embodiment 2

Cellobiose concentration and bacteriophage SP-lacY reproduction speed relationship are verified

1) culture of host S1030-AP1/AP2/MP LB culture medium is to logarithmic phase (0D₆₀₀=0.4).

2) wild type SP-lacY bacteriophage is diluted, is added in above-mentioned logarithmic phase host, makes its initial concentration in system 50pfu/mL。

3) cellobiose final concentration gradient be 29mM, 14.5mM, 2.9mM, 1.45mM, 0.29mM, 0.0029mM, 0.00029mM、0.00mM。

4) it is sampled for each cellobiose gradient every 15min, the bacteriophage SP-lacY in detection architecture is proliferated feelings Condition, as a result as shown in Figure 6.

In Fig. 6, the cellobiose of 0mM to 0.029mM concentration is essentially coincided with abscissa.

Fig. 6 experimental result shows that the growth rate of bacteriophage is directly proportional with the concentration of cellobiose, and it is dense to reduce cellobiose Degree, growth rate is slack-off, i.e., with the continuous reduction of cellobiose concentration during PACE evolves, LacY must be generated Front mutation improves it to the transfer efficiency of cellobiose to pack more front mutation SP-lacY filial generations, conversely, wild The SP-lacY of the type and SP-lacY being negatively mutated then constantly disappears in rounds of evolution and dilution.

Embodiment 3

The substrate specificity of PACE evolution LacY

1) host S1030-AP1/AP2/MP is cultivated to logarithmic phase (0D₆₀₀=0.4).

2) first round evolves, and in 1mL evolution system, initial fiber disaccharides final concentration 29mM, initial wild type SP-lacY are bitten Thallus 1.2 × 10⁵Pfu/mL, arabinose final concentration are always 1%, add host S1030-AP1/AP2/MP to 1mL, and 37 DEG C, 150RPM cultivates evolution 1h.Sampling, gradient dilution, 10 μ L of bacteriophage and 190 μ L logarithmic phase host S1030- after dilution AP1/AP2/MP mixing, 37 DEG C of placement 15min are mixed, with 50 DEG C of 0.5% soft agar 1mL containing 0.5% cellobiose It is even to be layered on diameter 60cm solid agar plate, 10min is stood, solidification is placed on 37 DEG C and is incubated overnight, the quantity of plaque is calculated, Determine the concentration of system pnagus medius.Single plaque is taken, with LacY gene piece on primer SP1-F, SP1-R amplification bacteriophage Section is sent to Hua Da sequencing, checks catastrophe.

3) the second wheel is evolved, and the 100 μ L of supernatant after taking first round evolution system to be centrifuged is as bacteriophage SP- to be evolved LacY (the mixing bacteriophage that bacteriophage at this time is wild type SP-lacY and its various mutant), cellobiose is final concentration of 14.5mM, arabinose final concentration are always 1%, add fresh host S1030-AP1/AP2/MP to 1mL, and 37 DEG C, 150RPM Cultivate evolution 1h.Sampling counting system pnagus medius concentration simultaneously detects catastrophe.

4) and so on, third round takes 100 μ L of the second wheel supernatant to continue to evolve, and reduces cellobiose concentration, fourth round Until last wheel is similarly.

5) during evolution, by calculating the concentration of every wheel system pnagus medius, the proliferation feelings for checking bacteriophage are seen Condition, if proliferation is very fast, next round can use last round of 10 μ L and execute Evolutionary experiments instead of 100 μ L；If being proliferated slower, wheel fibre It ties up under disaccharides concentration, increases wheel number of evolving.

6) with the quick reduction of cellobiose concentration, screening pressure becomes larger, the evolution disadvantage in time of 1h in mutation accumulation, 12nd wheel starts adjustment and evolves the time as 2h.

7) it evolves and executes 53 wheels altogether, cellobiose concentration is down to 100nM from 29mM.

8) it evolves as the result is shown (Fig. 7), when cellobiose concentration is higher than 29 μM, screening pressure is smaller, is nearly free from Mutation, further decrease cellobiose concentration, occur many mutational sites on LacY, wherein the mutational site A177V with into Change and carry out, obtains obvious accumulation, finally all mutant all include the mutation in the site A177V, illustrate this site to LacY The activity for transporting cellobiose is most important.

Embodiment 4

The verifying of evolution product LacYA177V transport activity

1) growth rate by comparison wild type SP-lacY and SP-lacYA177V under same cellobiose concentration, Verify evolution product LacYA177V cellobiose transport activity.

2) culture of host S1030-AP1/AP2/MP LB culture medium is to logarithmic phase (OD₆₀₀=0.4).

3) dilute wild type SP-lacY and and SP-lacYA177V bacteriophage to same concentration, be added separately to above-mentioned logarithm In phase host, final concentration of 290 μM or 58 μM of cellobiose.

4) 37 DEG C, 150rpm culture, sample, the bacteriophage multiplication situation in detection architecture every 15min.

5) whether comparison wild type SP-lacY and SP-lacYA177V is proliferated variant under same cellobiose concentration.

6) as the result is shown under (Fig. 8) same cellobiose concentration, SP-lacYA177V growth rate is apparently higher than wild type SP-lacY, illustrate the mutation of site A177V significantly improve LacY transhipment cellobiose activity, i.e., this set optimization after PACE evolutionary system can be for the activity for memebrane protein of evolving.

In addition, lactose transport protein is replaced with the transport protein of other carbohydrates or replaces with receptor protein, ion leads to Road albumen etc. can effectively realize the purpose that continuously-directional is evolved.

Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

SEQUENCE LISTING

<110>Shenzhen Xianjin Technology Academe

<120>the continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary

<130> 2018

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 5579

<212> DNA

<213>artificial sequence

<400> 1

atgcctctag attaattaat taagcggccg catcgatcgg gccctgaggc ctgcagggta 60

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cacacaggaa acagctatga aaaaattatt attcgcaatt cctttagttg ttcctttcta 180

ttctcactcc gctgaaactg ttcatcacca tcaccatcac gctgaaactg ttgaaagttg 240

tttagcaaaa ccccatacag aaaattcatt tactaacgtc tggaaagacg acaaaacttt 300

agatcgttac gctaactatg agggctgtct gtggaatgct acaggcgttg tagtttgtac 360

tggtgacgaa actcagtgtt acggtacatg ggttcctatt gggcttgcta tccctgaaaa 420

tgagggtggt ggctctgagg gtggcggttc tgagggtggc ggttctgagg gtggcggtac 480

taaacctcct gagtacggtg atacacctat tccgggctat acttatatca accctctcga 540

cggcacttat ccgcctggta ctgagcaaaa ccccgctaat cctaatcctt ctcttgagga 600

gtctcagcct cttaatactt tcatgtttca gaataatagg ttccgaaata ggcagggggc 660

attaactgtt tatacgggca ctgttactca aggcactgac cccgttaaaa cttattacca 720

gtacactcct gtatcatcaa aagccatgta tgacgcttac tggaacggta aattcagaga 780

ctgcgctttc cattctggct ttaatgagga tccattcgtt tgtgaatatc aaggccaatc 840

gtctgacctg cctcaacctc ctgtcaatgc tggcggcggc tctggtggtg gttctggtgg 900

cggctctgag ggtggtggct ctgagggtgg cggttctgag ggtggcggct ctgagggagg 960

cggttccggt ggtggctctg gttccggtga ttttgattat gaaaagatgg caaacgctaa 1020

taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg ctaaaggcaa 1080

acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg gtgacgtttc 1140

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tatttacttg gcgcgactaa aaaattgaat gtaggaactg ccgctattgt tcttcccaca 1680

gcccatccag tacgccaact tgaagatgtg aatttattgg atcaaatgtc aaaaggacga 1740

tttcggtttg gtatttgccg agggctttac aacaaggact ttcgcgtatt cggcacagat 1800

atgaataaca gtcgcgcctt agcggaatgc tggtacgggc tgataaagaa tggcatgaca 1860

gagggatata tggaagctga taatgaacat atcaagttcc ataaggtaaa agtaaacccc 1920

gcggcgtata gcagaggtgg cgcaccggtt tatgtggtgg ctgaatcagc ttcgacgact 1980

gagtgggctg ctcaatttgg cctaccgatg atattaagtt ggattataaa tactaacgaa 2040

aagaaagcac aacttgagct ttataatgaa gtggctcaag aatatgggca cgatattcat 2100

aatatcgacc attgcttatc atatataaca tctgtagatc atgactcaat taaagcgaaa 2160

gagatttgcc ggaaatttct ggggcattgg tatgattctt atgtgaatgc tacgactatt 2220

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gtattaaaag gacataaaga tactaatcgc cgtattgatt acagttacga aatcaatccc 2340

gtgggaacgc cgcaggaatg tattgacata attcaaaaag acattgatgc tacaggaata 2400

tcaaatattt gttgtggatt tgaagctaat ggaacagtag acgaaattat tgcttccatg 2460

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tatggcggtg gcggtagcgg cggtggcggt agcggcggtg gcggtagcgg cggtggcggt 2580

agcaaatttg gattgttctt ccttaacttc atcaattcaa caactgttca agaacagagt 2640

atagttcgca tgcaggaaat aacggagtat gttgataagt tgaattttga acagatttta 2700

gtgtatgaaa atcatttttc agataatggt gttgtcggcg ctcctctgac tgtttctggt 2760

tttctgctcg gtttaacaga gaaaattaaa attggttcat taaatcacat cattacaact 2820

catcatcctg tccgcatagc ggaggaagct tgcttattgg atcagttaag tgaagggaga 2880

tttattttag ggtttagtga ttgcgaaaaa aaagatgaaa tgcatttttt taatcgcccg 2940

gttgaatatc aacagcaact atttgaagag tgttatgaaa tcattaacga tgctttaaca 3000

acaggctatt gtaatccaga taacgatttt tatagcttcc ctaaaatatc tgtaaatccc 3060

catgcttata cgccaggcgg acctcggaaa tatgtaacag caaccagtca tcatattgtt 3120

gagtgggcgg ccaaaaaagg tattcctctc atctttaagt gggatgattc taatgatgtt 3180

agatatgaat atgctgaaag atataaagcc gttgcggata aatatgacgt tgacctatca 3240

gagatagacc atcagttaat gatattagtt aactataacg aagatagtaa taaagctaaa 3300

caagagacgc gtgcatttat tagtgattat gttcttgaaa tgcaccctaa tgaaaatttc 3360

gaaaataaac ttgaagaaat aattgcagaa aacgctgtcg gaaattatac ggagtgtata 3420

actgcggcta agttggcaat tgaaaagtgt ggtgcgaaaa gtgtattgct gtactttgaa 3480

ccaatgaatg atttgatgag ccaaaaaaat gtaatcaata ttgttgatga taatattaag 3540

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 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 (10)

1. a kind of plasmid, which is characterized in that including one or both of AP1 plasmid and AP2 plasmid；

AP1 plasmid carries gIII gene, and is provided with functional protein between the promoter and ribosome bind site of the plasmid Recognition site；

AP2 plasmid carrying function protein gene.

2. plasmid according to claim 1, which is characterized in that the promoter is J23109 promoter；

Preferably, the function egg that the functional protein recognition site in the AP1 is encoded by the functional protein gene of the AP2 plasmid White identification plays reptation behavior.

3. plasmid according to claim 1, which is characterized in that the gene order of the AP1 plasmid such as SEQ ID NO.1 institute Show, the gene order of the AP2 plasmid is as shown in SEQ ID NO.2.

The system 4. continuously-directional containing the bacteriophage of the described in any item AP1 plasmids of claim 1-3 and AP2 plasmid auxiliary is evolved System.

5. the continuously-directional evolutionary system of bacteriophage auxiliary according to claim 4, which is characterized in that the continuously-directional Evolutionary system further includes the bacteriophage of target gene replacement gIII gene, host strain, Mutagenic plasmid.

6. the continuously-directional evolutionary system of bacteriophage auxiliary according to claim 4, which is characterized in that claim 1-3 Described in any item AP1 plasmids and AP2 plasmid exist in the form of being transferred to host strain.

7. the continuously-directional evolutionary system of bacteriophage auxiliary according to claim 5, which is characterized in that the target gene Gene including memebrane protein；

Preferably, the memebrane protein includes transport protein, receptor protein, ionophorous protein.

8. according to the continuously-directional evolutionary system of the described in any item bacteriophage auxiliary of claim 5-7, which is characterized in that described Host strain is the Escherichia coli for carrying the F factor；The host strain is preferably E.coli S1030.

9. the directed evolution side carried out using the continuously-directional evolutionary system of the described in any item bacteriophage auxiliary of claim 5-8 Method, which is characterized in that AP1 plasmid, AP2 plasmid and Mutagenic plasmid are transferred in the host strain, obtain evolution host strain；

The bacteriophage of the evolution host strain and target gene replacement gIII gene is the case where screening pressure gradually becomes larger Culture screening is taken turns in lower progress more, obtains mutant.

10. directed evolution method according to claim 9, which is characterized in that the target gene is lactose transport protein Gene, the screening pressure are carried out by controlling the concentration of extracellular substrate cellobiose, and the screening pressure gradually becomes larger are as follows: born of the same parents Outer bottom fibres disaccharides concentration is gradually down to 29 μM or less from 29mM.