CN114317584A - Construction system of novel transposon mutant library, novel transposon mutant library and application - Google Patents

Abstract

The invention discloses a construction system of a novel transposon mutation library, which can transfer plasmids into a target strain by a chemical transformation method and an electric transformation method, then induce the expression of transposase, and only need a mutant strain containing transposon to obtain the transposon mutation library which almost covers the whole genome range. The construction system of the novel transposon library can not only increase the diversity of transposition mutation, but also is beneficial to determining the insertion position of the strain, and is beneficial to researching the genome annotation, functional analysis and drug resistance mechanism of pseudomonas aeruginosa.

Description

Construction system of novel transposon mutant library, novel transposon mutant library and application

Technical Field

The invention belongs to the technical field of gene mutation, and particularly relates to a construction system of a novel transposon mutant strain library, the novel transposon mutant library and application.

Background

Transposon (Tn), also known as a jumping gene, refers to a group of genetic elements capable of inserting a self gene into any one of the gene sequences in the genome, and is classified into a complex transposon and a complex transposon. The essence of the resistant transposon is a jumping gene system that binds the resistance gene within the element. The well-known transposon is Tn5, which encodes a gene for an aminoglycoside-resistant antibiotic such as kanamycin, neomycin; the Tn10 transposon is found to encode a tetracycline-resistant gene in some gram-negative bacteria, particularly in the enterobacteriaceae family.

The Insertion Sequence (IS) IS the simplest transfer element found in bacteria, and generally has the size of 600-2000 bp; the insertion sequence only codes transposase, the insertion sequence is the simplest transposon, and a gene sequence positioned between the two insertion sequences can also be transferred along with the insertion sequence; bacterial transposons, quinolone action target protection protein genes, insertion sequences can carry beta-lactamase genes, 16S rRNA methylase genes, aminoglycoside modification enzyme genes and efflux pump genes. In addition, it is possible to obtain highly active artificial transposons, such as sleepingBeauty, SB, by molecular reconstruction. Transposons such as SB and Mos1 have been widely used as gene transfer vectors in the fields of transgenosis, gene capture, gene therapy and the like, and have achieved good application effects.

In the early stage of the study, it was found that the insert ISRP10 blocked the P.aeruginosa oprD gene, which resulted in the resistance of the bacterium to imipenem (Qinghui Sun, et. al., Insertion sequence ISRP10 inactivation of the OPrD gene inimiphene-resistant Pseudomonas aeruginosa viral polysaccharides [ J ]. International Journal of antimicrobial agents.2016,5(47):375-379.), and that the positive rate of the insert was 32.8% as measured by its positive rate. By analyzing that ISRP10 belongs to IS30 family, the length IS 1115-1117bp, and the content of G + C IS 60.22%. Amino acids 174-327 of the insertion sequence ISRP10 code for integrase which has nuclease activity, exonuclease activity, hydrolase activity, DNA recombination function and DNA transfer function. There are also two metal active center positions 184aa and 241aa, respectively, in the catalytic domain, and both are magnesium ion active centers, as shown in fig. 1.

The pseudomonas aeruginosa is clinically important pathogenic bacteria, the pseudomonas aeruginosa can not only show natural drug resistance to various antibacterial drugs, but also obtain drug resistance through antibiotic induction, and the drug resistance of the pseudomonas aeruginosa is more serious along with the wide application of the antibacterial drugs, so that the infection of the pseudomonas aeruginosa is difficult to treat clinically. The mechanism of drug resistance generation of pseudomonas aeruginosa is very complex, at present, transposons can be used for research in order to research the drug resistance mechanism of pseudomonas aeruginosa, but the current transposon library system is generally constructed by adopting a conjugal transfer mode, but the transposon library constructed by the method has the defects of low efficiency, complex operation, small library capacity and the like.

In order to solve the problems existing in the current library construction and enable the transposon to be widely used, the invention provides a novel construction system of a transposon mutation library, plasmids can be transferred into a target strain through a chemical transformation method and an electric transformation method, then the expression of transposase is induced, and the transposon mutation library almost covering the whole genome range can be obtained only by a mutant strain containing the transposon. The construction system of the novel transposon library can not only increase the diversity of transposition mutation, but also is beneficial to determining the insertion position of the strain, and is beneficial to researching the genome annotation, functional analysis and drug resistance mechanism of pseudomonas aeruginosa.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a construction system of a novel transposon mutant strain library, the novel transposon mutant strain library and application.

The technical scheme adopted by the invention for solving the technical problem is as follows:

a construction system of a novel transposon mutant strain library comprises the following construction steps:

(1) imitating a natural transposon plasmid, artificially constructing a novel transposon plasmid constructed by taking pUCP20 as a framework, wherein the plasmid replaces a LacZ alpha region of pUCP20 with a novel transposable region, and the transposon region comprises transposase ISRP10 regulated and controlled by Plac, inverted terminal repeat ITRs and a gentamicin resistance gene;

(2) adding a transposase gene RP10 which can be regulated by a Lac promoter gene into the novel transposable region;

(3) directly carrying out electrotransformation on the recombinant plasmid and introducing the recombinant plasmid into a receptor cell, and then inducing transposase expression by IPTG to carry out transposition jumping;

(4) the transposon mutant has the possibility of containing a novel transposable region at any position in the genome, and the transposon region comprises: a transposase ISRP10 regulated by Plac, terminal repeat ITRs and a gentamicin resistance gene;

(5) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹And (3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM to induce the expression of the transposase when cfu/mL is reached, carrying out induced culture for 2 hours to enable the transposon to jump, and randomly obtaining a transposon mutant strain library with various inserted gene forms in the whole genome range.

Further, the construction steps are as follows:

(1) imitating a natural transposon plasmid, and artificially constructing a novel transposon plasmid vector which replaces a LacZ alpha gene in a pUCP20 plasmid by a novel transposable region on the basis of the pUCP20 transposon plasmid;

(2) the transposase RP10 of the novel transposable region can be regulated and controlled by Lac promoter genes;

(3) firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK through an electrochemical conversion method to obtain a transposon mutant strain containing transposons of different species;

(4) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹At cfu/mL, IPTG was added to the final concentration of 1mM, and induction culture was carried out for 2 hours to allow transposon jumping, thereby randomly obtaining a transposon mutant library of various inserted gene forms in the whole genome.

Further, the nucleotide sequence of the pUCP20 transposon plasmid is SEQ ID NO. 1.

Further, the nucleotide sequence of the pUCP20 transposon plasmid with the LacZ α gene removed in the first step is SEQ ID No. 2.

Further, the nucleotide sequence of the insertion sequence ISRP10 is SEQ ID NO. 3.

Further, the nucleotide sequence of the novel transposable region in the first step is SEQ ID No. 4.

Further, the nucleotide sequence of the novel transposon plasmid vector in the step is SEQ ID No. 5.

Further, the concentration and the volume of the transposon mutant strain can be controlled according to the requirement for constructing the library, and the process of inducing transposon jumping by the transposase can be regulated and controlled.

A novel transposon mutation library obtained by using the construction system as described above.

The construction system is applied to the construction of the pseudomonas aeruginosa insertion mutant strain library.

The beneficial effects obtained by the invention are as follows:

1. construction of transposon mutant libraries is now widely used for functional annotation and functional analysis of genomes. At present, a transposable library system is generally constructed in a joint transfer mode, but the efficiency of the transposable library constructed by the method is low and can only reach 10^-3-10^-4Moreover, the resulting library is of a small capacity and cannot cover the entire genome of the bacteria. The invention constructs a novel transposon mutation library system by utilizing the advantage that the pUCP20 plasmid has wide host replication initiation region. The insertion sequence ISRP10 is firstly transformed and cloned into a pUCP20 plasmid to enable the expression of the ISRP10 to be regulated and controlled by IPTG, and a gentamicin resistance gene is additionally added to assist screening. A transposon-containing mutant is obtained through electrotransformation, the mutant is taken as a starting strain, a novel transposase RP10 is induced to express under the induction of IPTG, transposons are jumped and inserted into different genes, and a transposon mutation library with various insertion gene forms is obtained. The construction system of the novel transposon library can not only increase the diversity of transposition mutations, but also help to determine the insertion position of the strain.

2. The construction system can systematically and efficiently obtain the pseudomonas aeruginosa transposon mutant strain library, and is beneficial to the research of the pseudomonas aeruginosa drug resistance mechanism.

Drawings

FIG. 1 is a diagram of an integrase assay for ISRP10 according to the invention;

FIG. 2 is a genetic map of the vector plasmid pUCP2021 of the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A construction system of a novel transposon mutant strain library comprises the following construction steps:

(1) artificially constructing a novel transposon plasmid constructed by using pUCP20 as a framework by simulating a natural transposon plasmid, wherein the novel transposon plasmid replaces a LacZ alpha region of pUCP20 with a novel transposable region, and the transposon region comprises transposase ISRP10 regulated and controlled by Plac, Inverted Terminal Repeat (ITRs) and gentamicin resistance genes;

(2) adding a transposase gene RP10 which can be regulated by a Lac promoter gene into the novel transposable region;

(3) directly carrying out electrotransformation on the recombinant plasmid and introducing the recombinant plasmid into a receptor cell, and then inducing transposase expression by IPTG to carry out transposition jumping;

(4) the transposon mutant has the possibility of containing a novel transposable region at any position in the genome, and the transposon region comprises: the transposase ISRP10, terminal repeat ITRs (ITRs) and gentamicin resistance genes regulated by Plac;

(5) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹And (3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM to induce the expression of the transposase when cfu/mL is reached, carrying out induced culture for 2 hours to enable the transposon to jump, and randomly obtaining a transposon mutant strain library with various inserted gene forms in the whole genome range.

Preferably, the construction steps are as follows:

(1) imitating a natural transposon plasmid, and artificially constructing a novel transposon plasmid vector which replaces a LacZ alpha gene in a pUCP20 plasmid by a novel transposable region on the basis of the pUCP20 transposon plasmid, wherein the genetic map of the novel transposon plasmid vector is shown in figure 2;

(2) the transposase RP10 of the novel transposable region can be regulated and controlled by Lac promoter genes;

(3) firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK through an electrochemical conversion method to obtain a transposon mutant strain containing transposons of different species; the transposon mutant strain is pseudomonas aeruginosa containing a transposase gene RP10 which is stably expressed and regulated by Lac;

(4) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹At cfu/mL, IPTG was added to the final concentration of 1mM, and induction culture was carried out for 2 hours to allow transposon jumping, thereby randomly obtaining a transposon mutant library of various inserted gene forms in the whole genome.

Preferably, the nucleotide sequence of the pUCP20 transposon plasmid is SEQ ID NO. 1.

The transposon plasmid vector has the following characteristics:

removing the gene sequence of pUCP20 transposon plasmid in the LacZ alpha gene region;

adding a pre-constructed novel transposon area in the original LacZ alpha gene area, wherein the transposon area comprises: the Plac regulated insert ISRP10, terminal repeats (ITRs) and the gentamicin resistance gene;

preferably, the nucleotide sequence of the pUCP20 transposon plasmid with the LacZ α gene removed in the first step is SEQ ID No. 2.

Preferably, the nucleotide sequence of the insertion sequence ISRP10 is SEQ ID NO. 3.

Preferably, the nucleotide sequence of the novel transposable region in the first step is SEQ ID No. 4.

Preferably, the nucleotide sequence of the novel transposon plasmid vector of the step is SEQ ID No. 5.

Preferably, the concentration and the volume of the transposon mutant strain can be controlled according to the requirement for constructing the library, and the process of inducing transposon jumping by the transposase can be regulated and controlled.

A novel transposon mutation library obtained by using the construction system as described above.

The construction system is applied to the construction of the pseudomonas aeruginosa insertion mutant strain library.

Specifically, the preparation and detection are as follows:

the vector of the invention takes a pUCP20 plasmid preserved in a laboratory as a starting plasmid, firstly removes a LacZ alpha gene of the pUCP20 plasmid, and then adds a novel transposable region in the region, wherein the transposable region comprises: the Plac-regulated insert sequence ISRP10, terminal repeats (ITRs) and the gentamicin resistance gene. Under the induction of IPTG, the transposon can carry an insertion sequence ISRP10 gene regulated by Plac and a Gm resistance gene to jump into a new recipient bacterium genome together, and the transformed transposition plasmid is named as pUCP 2021. Electrochemically transferring the pUCP2021 plasmid into PAK to obtain a donor strain containing transposase genes RP10 and Gm resistance genes regulated by IPTG; when a recipient strain containing a transposon is cultured alone, a variety of recipient strains in the form of an inserted gene can be obtained by adding IPTG for induction.

The construction process of the transposon plasmid and the construction process of the novel transposon library of the present invention are described in detail below.

Construction of transposon plasmid (pUCP2021)

Firstly, designing a primer for amplification to obtain an insertion sequence ISRP10 (wherein the coding gene sequence of the insertion sequence ISRP10 is SEQ ID NO.3)

Clinical pseudomonas aeruginosa TJ173 (the oprD gene of the strain has an insert sequence ISRP10) is taken as a template, and an upstream primer is utilized: 5'-ATCTCGCAGGTTGGGTATGTTCATGCT-3' and the downstream primer: 5'-CCACCATTCAAATCGGTCGTACCCAAG-3' amplification to obtain the insertion sequence ISRP10 gene.

In the second step, the Gm resistance encoding gene sequence was derived from the laboratory-conserved plasmid pEX18 Gm.

Third, the LacZ alpha gene (SEQ ID NO.2) was removed by storing plasmid pUCP20 in the laboratory.

Fourthly, adding Gm resistance of pEX18Gm plasmid at one end of ITRs of the gene coded by the insertion sequence ISRP10 obtained by PCR, controlling the expression of the novel transposase RP10 by using a Plac promoter of pUCP20, reconstructing a novel transposase region and a pUCP20 framework with the LacZ alpha gene removed to obtain a novel transposon plasmid vector pUCP2021 (the nucleotide sequence is SEQ ID NO.5)

This step was handed to Jinzhi sequencing, Suzhou, China for sequence synthesis.

And fifthly, successfully and electrically transforming the novel transposon plasmid vector pUCP2021 into the strain PAK to obtain a recombinant strain PAK/pUCP2021, namely a novel transposition system strain, wherein the strain can generate transposase through IPTG induction to enable transposons to jump to obtain a transposable library which covers the whole gene range and has multiple types and large quantity.

Second, novel construction system of transposable library

Firstly, the constructed plasmid pUCP2021 is electrically transferred into a target strain PAK, and a conversion solution is coatedCloth on Amp¹⁰⁰、Gm¹⁰⁰The plasmid-containing recipient strain was obtained by overnight culture on LB medium (Takara Shuzo).

And secondly, verifying whether the target plasmid is contained in the recipient strain or not by using the quality-improving particles, and if the verification is correct, using the strain as a starting strain in the next step.

Thirdly, streaking and purifying the strain containing the plasmid three times, and culturing overnight, wherein the antibiotic concentration is Amp¹⁰⁰And Gm¹⁰⁰。

The fourth step, the overnight cultured strain was transferred at 3% transfer amount and cultured to OD₆₀₀When the final concentration was 0.6, IPTG was added to the final concentration of 1mM to induce transposase expression, and a whole genome transposon library containing various gene insertions was obtained.

Specifically, the method comprises the following steps:

1. the constructed pUCP2021 plasmid is electrically transferred to the recipient bacterium pseudomonas aeruginosa PAK.

2. Coating the conversion solution on Amp¹⁰⁰And Gm¹⁰⁰The plasmid-containing recipient strain was obtained by culturing the cells in LB medium (Takara Shuzo) at 37 ℃ overnight for 12 hours.

4. And selecting 10 transformants for quality improvement particle verification, wherein 10 strains only have No.3 strain as a negative result.

5. Taking an SQ1 strain of 10 strains as an original strain, transferring the strain to the overnight-cultured SQ1 with the transfer amount of 3 percent, and culturing the strain to OD₆₀₀When the total gene content is 0.6, IPTG with the final concentration of 1mM is added to induce the expression of transposase, and the transposase is induced and cultured for 2h, so that the transposase is expressed to jump the transposons to other positions of the PAK strain genome, and a whole genome transposon library of various gene insertion forms covering the whole gene is obtained.

Seq1

Nucleotide sequence of pUCP20 transposon plasmid

Seq2

Nucleotide sequence of pUCP20 transposon plasmid with LacZ alpha gene removed

Seq3.

Nucleotide sequence of insertion sequence ISRP10 coding gene

Seq4.

Nucleotide sequence of gene encoding novel transposable region (RP10)

Seq5.

Nucleotide sequence of novel transposon plasmid vector

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Sequence listing

<110> Hainan college of medicine, Tianjin university of science and technology

<120> construction system of novel transposon mutant library, novel transposon mutant library and use

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3643

<212> DNA/RNA

<213> nucleotide sequence of pUCP20 transposon plasmid (Unknown)

<400> 1

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctgcc 2220

agatccagcg gcatctgggt tagtcgagcg cgggccgctt cccatgtctc accagggcga 2280

gcctgtttcg cgatctcagc atctgaaatc ttcccggcct tgcgcttcgc tggggcctta 2340

cccaccgcct tggcgggctt cttcggtcca aaactgaaca acagatgtgt gaccttgcgc 2400

ccggtctttc gctgcgccca ctccacctgt agcgggctgt gctcgttgat ctgcgtcacg 2460

gctggatcaa gcactcgcaa cttgaagtcc ttgatcgagg gataccggcc ttccagttga 2520

aaccactttc gcagctggtc aatttctatt tcgcgctggc cgatgctgtc ccattgcatg 2580

agcagctcgt aaagcctgat cgcgtgggtg ctgtccatct tggccacgtc agccaaggcg 2640

tatttggtga actgtttggt gagttccgtc aggtacggca gcatgtcttt ggtgaacctg 2700

agttctacac ggccctcacc ctcccggtag atgattgttt gcacccagcc ggtaatcatc 2760

acactcggtc ttttcccctt gccattgggc tcttgggtta accggacttc ccgccgtttc 2820

aggcgcaggg ccgcttcttt gagctggttg taggaagatt cgatagggac acccgccatc 2880

gtcgctatgt cctccgccgt cactgaatac atcacttcat cggtgacagg ctcgctcctc 2940

ttcacctggc taatacaggc cagaacgatc cgctgttcct gaacactgag gcgatacgcg 3000

gcctcgacca gggcattgct tttgtaaacc attgggggtg aggccacgtt cgacattcct 3060

tgtgtataag gggacactgt atctgcgtcc cacaatacaa caaatccgtc cctttacaac 3120

aacaaatccg tcccttctta acaacaaatc cgtcccttaa tggcaacaaa tccgtccctt 3180

tttaaactct acaggccacg gattacgtgg cctgtagacg tcctaaaagg tttaaaaggg 3240

aaaaggaaga aaagggtgga aacgcaaaaa acgcaccact acgtggcccc gttggggccg 3300

catttgtgcc cctgaagggg cgggggaggc gtctgggcaa tccccgtttt accagtcccc 3360

tatcgccgcc tgagagggcg caggaagcga gtaatcaggg tatcgaggcg gattcaccct 3420

tggcgtccaa ccagcggcac cagcggcgcc tgagaggtat ggtgcactct cagtacaatc 3480

tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 3540

tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 3600

tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga 3643

<210> 2

<211> 3643

<212> DNA/RNA

<213> removing the nucleotide sequence of pucp20 transposon plasmid (Unknown) of lacz. alpha. Gene

<400> 2

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctgcc 2220

agatccagcg gcatctgggt tagtcgagcg cgggccgctt cccatgtctc accagggcga 2280

gcctgtttcg cgatctcagc atctgaaatc ttcccggcct tgcgcttcgc tggggcctta 2340

cccaccgcct tggcgggctt cttcggtcca aaactgaaca acagatgtgt gaccttgcgc 2400

ccggtctttc gctgcgccca ctccacctgt agcgggctgt gctcgttgat ctgcgtcacg 2460

gctggatcaa gcactcgcaa cttgaagtcc ttgatcgagg gataccggcc ttccagttga 2520

aaccactttc gcagctggtc aatttctatt tcgcgctggc cgatgctgtc ccattgcatg 2580

agcagctcgt aaagcctgat cgcgtgggtg ctgtccatct tggccacgtc agccaaggcg 2640

tatttggtga actgtttggt gagttccgtc aggtacggca gcatgtcttt ggtgaacctg 2700

agttctacac ggccctcacc ctcccggtag atgattgttt gcacccagcc ggtaatcatc 2760

acactcggtc ttttcccctt gccattgggc tcttgggtta accggacttc ccgccgtttc 2820

aggcgcaggg ccgcttcttt gagctggttg taggaagatt cgatagggac acccgccatc 2880

gtcgctatgt cctccgccgt cactgaatac atcacttcat cggtgacagg ctcgctcctc 2940

ttcacctggc taatacaggc cagaacgatc cgctgttcct gaacactgag gcgatacgcg 3000

gcctcgacca gggcattgct tttgtaaacc attgggggtg aggccacgtt cgacattcct 3060

tgtgtataag gggacactgt atctgcgtcc cacaatacaa caaatccgtc cctttacaac 3120

aacaaatccg tcccttctta acaacaaatc cgtcccttaa tggcaacaaa tccgtccctt 3180

tttaaactct acaggccacg gattacgtgg cctgtagacg tcctaaaagg tttaaaaggg 3240

aaaaggaaga aaagggtgga aacgcaaaaa acgcaccact acgtggcccc gttggggccg 3300

catttgtgcc cctgaagggg cgggggaggc gtctgggcaa tccccgtttt accagtcccc 3360

tatcgccgcc tgagagggcg caggaagcga gtaatcaggg tatcgaggcg gattcaccct 3420

tggcgtccaa ccagcggcac cagcggcgcc tgagaggtat ggtgcactct cagtacaatc 3480

tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 3540

tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 3600

tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga 3643

<210> 3

<211> 1029

<212> DNA/RNA

<213> nucleotide sequence of Gene encoding iprp 10 inserted sequence (Unknown)

<400> 3

atgtcttata ccgaactcag cgttgaagag cgcgccacca ttcaaatcgg tcgtacccaa 60

ggcttcagcc tgcgcaggat tgcctgcttg atcaaccgat ccccttcgac catcagccgt 120

gagctgcgcc gtaaccgagg tgcttgcggt ggctactcgg cccgcctggc ccagcagcaa 180

atgcaggccc gccgccaggt ttgtcgaccg atgcgaaaac tgttgccggg tagcgagcgc 240

ttcgaactgg tgacccatat gctgcgtgag cgtttgtctc ccgagcagat tgccggcaag 300

ctgcgcagca tgaacatacc taacctgcga gatgcctacg tctgtcgcga gacgatctac 360

aacgcgatct atgccctgcc agtgggtgag ctgcgtaagg agctgatcat ctgtctgcgc 420

caaggcaaga cgacgcgcag gccgcgctct ggtggcgtgg atcggcgcgg ccagatcccc 480

gagatggtca gtattcatgt gcgcccgccg gagattgaag acaggctgat gccggggcat 540

tgggaaggcg acctcatcaa gggtaaggcc aacgcctcgt ccgtcggtac gctggtggaa 600

cgacccagtg gctacctgat gttggtgaag atgaacgacg cgacggcgac ctcggcgctg 660

gaaggcttca gcgcagcgct caatagcatg ccgctggaga tgcgcaagag catgacttac 720

gaccagggcc gggagatggc gcgacacgcc gagatcaccc aaagaaccgg cgtggcgatc 780

tacttctgcg acccgcacag cccctggcag cgcggcagca acgaaaacat caacggcctg 840

attcgccagt acttgcccaa gggcacagac ctgtcggtac atagccagga ggagctggac 900

gccattgcgc tgcaactgaa catgcgaccg cgtaagcgct tcgacttcaa atgcccaatc 960

gaagttatgg acgaggtgat gcagaaggcc atggctatgc ggcatgatgc tccagtttca 1020

attcaatga 1029

<210> 4

<211> 2082

<212> DNA/RNA

<213> novel transposable region (nucleotide sequence Unknown of rp 10-encoding gene)

<400> 4

ggcggcggca aaaactgagt gcaacacgag cgcccaatac gcaaaccgcc tctccccgcg 60

cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 120

gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 180

atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 240

agctatgtct tataccgaac tcagcgttga agagcgcgcc accattcaaa tcggtcgtac 300

ccaaggcttc agcctgcgca ggattgcctg cttgatcaac cgatcccctt cgaccatcag 360

ccgtgagctg cgccgtaacc gaggtgcttg cggtggctac tcggcccgcc tggcccagca 420

gcaaatgcag gcccgccgcc aggtttgtcg accgatgcga aaactgttgc cgggtagcga 480

gcgcttcgaa ctggtgaccc atatgctgcg tgagcgtttg tctcccgagc agattgccgg 540

caagctgcgc agcatgaaca tacctaacct gcgagatgcc tacgtctgtc gcgagacgat 600

ctacaacgcg atctatgccc tgccagtggg tgagctgcgt aaggagctga tcatctgtct 660

gcgccaaggc aagacgacgc gcaggccgcg ctctggtggc gtggatcggc gcggccagat 720

ccccgagatg gtcagtattc atgtgcgccc gccggagatt gaagacaggc tgatgccggg 780

gcattgggaa ggcgacctca tcaagggtaa ggccaacgcc tcgtccgtcg gtacgctggt 840

ggaacgaccc agtggctacc tgatgttggt gaagatgaac gacgcgacgg cgacctcggc 900

gctggaaggc ttcagcgcag cgctcaatag catgccgctg gagatgcgca agagcatgac 960

ttacgaccag ggccgggaga tggcgcgaca cgccgagatc acccaaagaa ccggcgtggc 1020

gatctacttc tgcgacccgc acagcccctg gcagcgcggc agcaacgaaa acatcaacgg 1080

cctgattcgc cagtacttgc ccaagggcac agacctgtcg gtacatagcc aggaggagct 1140

ggacgccatt gcgctgcaac tgaacatgcg accgcgtaag cgcttcgact tcaaatgccc 1200

aatcgaagtt atggacgagg tgatgcagaa ggccatggct atgcggcatg atgctccagt 1260

ttcaattcaa tgagaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 1320

aatcgatgcg aattggccgc ggcgttgtga caatttaccg aacaactccg cggccgggaa 1380

gccgatctcg gcttgaacga attgttaggt ggcggtactt gggtcgatat caaagtgcat 1440

cacttcttcc cgtatgccca actttgtata gagagccact gcgggatcgt caccgtaatc 1500

tgcttgcacg tagatcacat aagcaccaag cgcgttggcc tcatgcttga ggagattgat 1560

gagcgcggtg gcaatgccct gcctccggtg ctcgccggag actgcgagat catagatata 1620

gatctcacta cgcggctgct caaacctggg cagaacgtaa gccgcgagag cgccaacaac 1680

cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta ctacggagca agttcccgag 1740

gtaatcggag tccggctgat gttgggagta ggtggctacg tctccgaact cacgaccgaa 1800

aagatcaaga gcagcccgca tggatttgac ttggtcaggg ccgagcctac atgtgcgaat 1860

gatgcccata cttgagccac ctaactttgt tttagggcga ctgccctgct gcgtaacatc 1920

gttgctgctg cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac 1980

gcgcttgctg cttggatgcc cgaggcatag actgtacaaa aaaacagtca taacaagcca 2040

tgaaaaccgc cactggtgtt gcactcagct cctgcaaccg cc 2082

<210> 5

<211> 5470

<212> DNA/RNA

<213> nucleotide sequence of novel transposon plasmid vector (Unknown)

<400> 5

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gaggcggcgg caaaaactga gtgcaacacg agcgcccaat 1980

acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 2040

tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 2100

ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 2160

ataacaattt cacacaggaa acagctatgt cttataccga actcagcgtt gaagagcgcg 2220

ccaccattca aatcggtcgt acccaaggct tcagcctgcg caggattgcc tgcttgatca 2280

accgatcccc ttcgaccatc agccgtgagc tgcgccgtaa ccgaggtgct tgcggtggct 2340

actcggcccg cctggcccag cagcaaatgc aggcccgccg ccaggtttgt cgaccgatgc 2400

gaaaactgtt gccgggtagc gagcgcttcg aactggtgac ccatatgctg cgtgagcgtt 2460

tgtctcccga gcagattgcc ggcaagctgc gcagcatgaa catacctaac ctgcgagatg 2520

cctacgtctg tcgcgagacg atctacaacg cgatctatgc cctgccagtg ggtgagctgc 2580

gtaaggagct gatcatctgt ctgcgccaag gcaagacgac gcgcaggccg cgctctggtg 2640

gcgtggatcg gcgcggccag atccccgaga tggtcagtat tcatgtgcgc ccgccggaga 2700

ttgaagacag gctgatgccg gggcattggg aaggcgacct catcaagggt aaggccaacg 2760

cctcgtccgt cggtacgctg gtggaacgac ccagtggcta cctgatgttg gtgaagatga 2820

acgacgcgac ggcgacctcg gcgctggaag gcttcagcgc agcgctcaat agcatgccgc 2880

tggagatgcg caagagcatg acttacgacc agggccggga gatggcgcga cacgccgaga 2940

tcacccaaag aaccggcgtg gcgatctact tctgcgaccc gcacagcccc tggcagcgcg 3000

gcagcaacga aaacatcaac ggcctgattc gccagtactt gcccaagggc acagacctgt 3060

cggtacatag ccaggaggag ctggacgcca ttgcgctgca actgaacatg cgaccgcgta 3120

agcgcttcga cttcaaatgc ccaatcgaag ttatggacga ggtgatgcag aaggccatgg 3180

ctatgcggca tgatgctcca gtttcaattc aatgagaagt tttaaatcaa tctaaagtat 3240

atatgagtaa acttggtctg acaatcgatg cgaattggcc gcggcgttgt gacaatttac 3300

cgaacaactc cgcggccggg aagccgatct cggcttgaac gaattgttag gtggcggtac 3360

ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta tagagagcca 3420

ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac ataagcacca agcgcgttgg 3480

cctcatgctt gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg tgctcgccgg 3540

agactgcgag atcatagata tagatctcac tacgcggctg ctcaaacctg ggcagaacgt 3600

aagccgcgag agcgccaaca accgcttctt ggtcgaaggc agcaagcgcg atgaatgtct 3660

tactacggag caagttcccg aggtaatcgg agtccggctg atgttgggag taggtggcta 3720

cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg catggatttg acttggtcag 3780

ggccgagcct acatgtgcga atgatgccca tacttgagcc acctaacttt gttttagggc 3840

gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat cgttgctgct ccataacatc 3900

aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat agactgtaca 3960

aaaaaacagt cataacaagc catgaaaacc gccactggtg ttgcactcag ctcctgcaac 4020

cgccctccag tttcaattca atgagccaga tccagcggca tctgggttag tcgagcgcgg 4080

gccgcttccc atgtctcacc agggcgagcc tgtttcgcga tctcagcatc tgaaatcttc 4140

ccggccttgc gcttcgctgg ggccttaccc accgccttgg cgggcttctt cggtccaaaa 4200

ctgaacaaca gatgtgtgac cttgcgcccg gtctttcgct gcgcccactc cacctgtagc 4260

gggctgtgct cgttgatctg cgtcacggct ggatcaagca ctcgcaactt gaagtccttg 4320

atcgagggat accggccttc cagttgaaac cactttcgca gctggtcaat ttctatttcg 4380

cgctggccga tgctgtccca ttgcatgagc agctcgtaaa gcctgatcgc gtgggtgctg 4440

tccatcttgg ccacgtcagc caaggcgtat ttggtgaact gtttggtgag ttccgtcagg 4500

tacggcagca tgtctttggt gaacctgagt tctacacggc cctcaccctc ccggtagatg 4560

attgtttgca cccagccggt aatcatcaca ctcggtcttt tccccttgcc attgggctct 4620

tgggttaacc ggacttcccg ccgtttcagg cgcagggccg cttctttgag ctggttgtag 4680

gaagattcga tagggacacc cgccatcgtc gctatgtcct ccgccgtcac tgaatacatc 4740

acttcatcgg tgacaggctc gctcctcttc acctggctaa tacaggccag aacgatccgc 4800

tgttcctgaa cactgaggcg atacgcggcc tcgaccaggg cattgctttt gtaaaccatt 4860

gggggtgagg ccacgttcga cattccttgt gtataagggg acactgtatc tgcgtcccac 4920

aatacaacaa atccgtccct ttacaacaac aaatccgtcc cttcttaaca acaaatccgt 4980

cccttaatgg caacaaatcc gtcccttttt aaactctaca ggccacggat tacgtggcct 5040

gtagacgtcc taaaaggttt aaaagggaaa aggaagaaaa gggtggaaac gcaaaaaacg 5100

caccactacg tggccccgtt ggggccgcat ttgtgcccct gaaggggcgg gggaggcgtc 5160

tgggcaatcc ccgttttacc agtcccctat cgccgcctga gagggcgcag gaagcgagta 5220

atcagggtat cgaggcggat tcacccttgg cgtccaacca gcggcaccag cggcgcctga 5280

gaggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 5340

cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 5400

agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 5460

aaacgcgcga 5470

Claims (10)

1. A construction system of a novel transposon mutant library, which is characterized in that: the construction steps are as follows:

(1) imitating a natural transposon plasmid, artificially constructing a novel transposon plasmid constructed by taking pUCP20 as a framework, wherein the plasmid replaces a LacZ alpha region of pUCP20 with a novel transposable region, and the transposon region comprises transposase ISRP10 regulated and controlled by Plac, inverted terminal repeat ITRs and a gentamicin resistance gene;

(2) adding a transposase gene RP10 which can be regulated by a Lac promoter gene into the novel transposable region;

(3) directly carrying out electrotransformation on the recombinant plasmid and introducing the recombinant plasmid into a receptor cell, and then inducing transposase expression by IPTG to carry out transposition jumping;

(4) the transposon mutant has the possibility of containing a novel transposable region at any position in the genome, and the transposon region comprises: a transposase ISRP10 regulated by Plac, terminal repeat ITRs and a gentamicin resistance gene;

(5) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹And (3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM to induce the expression of the transposase when cfu/mL is reached, carrying out induced culture for 2 hours to enable the transposon to jump, and randomly obtaining a transposon mutant strain library with various inserted gene forms in the whole genome range.

2. The system for constructing a novel transposon mutant library as claimed in claim 1, wherein: the construction steps are as follows:

(1) imitating a natural transposon plasmid, and artificially constructing a novel transposon plasmid vector which replaces a LacZ alpha gene in a pUCP20 plasmid by a novel transposable region on the basis of the pUCP20 transposon plasmid;

(2) the transposase RP10 of the novel transposable region can be regulated and controlled by Lac promoter genes;

(3) firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK through an electrochemical conversion method to obtain a transposon mutant strain containing transposons of different species;

(4) amplifying and culturing a transposon mutant strain, and obtaining 10 cells¹¹At cfu/mL, IPTG was added to the final concentration of 1mM, and induction culture was carried out for 2 hours to allow transposon jumping, thereby randomly obtaining a transposon mutant library of various inserted gene forms in the whole genome.

3. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: the nucleotide sequence of the pUCP20 transposon plasmid is SEQ ID NO. 1.

4. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: the nucleotide sequence of the pUCP20 transposon plasmid with the LacZ alpha gene removed in the step is SEQ ID NO. 2.

5. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: the nucleotide sequence of the insertion sequence ISRP10 is SEQ ID NO. 3.

6. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: the nucleotide sequence of the novel transposition subregion in the step is SEQ ID NO. 4.

7. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: the nucleotide sequence of the novel transposon plasmid vector in the step is SEQ ID NO. 5.

8. The system for constructing a novel transposon mutant library as claimed in claim 1 or 2, wherein: in the step fifthly, the bacterial concentration and the volume of the transposon mutant strain can be controlled according to the requirement of constructing the library, and the process of inducing transposon jumping by the transposase can be regulated and controlled.

9. A novel transposon mutation library obtained by using the construction system according to any one of claims 1 to 8.

10. Use of a construction system according to any one of claims 1 to 8 for the construction of a library of inserted mutants of pseudomonas aeruginosa.

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