CN114317584B - Construction system of novel transposon mutant strain library, novel transposon mutant library and application - Google Patents
Construction system of novel transposon mutant strain library, novel transposon mutant library and application Download PDFInfo
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- CN114317584B CN114317584B CN202111394427.0A CN202111394427A CN114317584B CN 114317584 B CN114317584 B CN 114317584B CN 202111394427 A CN202111394427 A CN 202111394427A CN 114317584 B CN114317584 B CN 114317584B
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Abstract
The invention discloses a novel construction system of transposon mutation library, which can transfer plasmids into target strains through chemical transformation and electric transformation methods, then induce expression of transposase, and can obtain transposon mutation library almost covering the whole genome range by only one mutant strain containing transposon. The construction system of the novel transposon library not only can increase the diversity of transposition mutations, but also is beneficial to determining the insertion position of strains and researching genome annotation, functional analysis and drug resistance mechanism of pseudomonas aeruginosa.
Description
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
Transposons (Tn), also known as skip genes, refer to a group of genetic elements that insert their own genes into any one of the genetic sequences in the genome, and are classified into complex transposons and complex transposons. The nature of the resistant transposon is a jump gene system that binds the resistance gene within the element. The more well-known transposon is Tn5, which encodes a gene for an anti-aminoglycoside antibiotic such as kanamycin, neomycin; in some gram-negative bacteria, the Tn10 transposon is found to encode a tetracycline-resistant gene, particularly in the Enterobacteriaceae family.
The insertion sequence (Insertion sequence, IS) IS the simplest class of transfer elements found in bacteria, typically in the size range 600-2000 bp; the insertion sequence only codes for a transposase, the insertion sequence is the simplest transposon, and the gene sequence between two insertion sequences can also be transferred with the insertion sequences; the bacterial transposon and quinolone acting target protecting protein gene and the inserted sequence can carry beta-lactamase gene, 16S rRNA methylase gene, aminoglycoside modifying enzyme gene and efflux pump gene. In addition, highly active artificial transposons, such as SleepingBeauty, SB, can be obtained by molecular reconstruction. Transposons such as SB and Mos1 are widely used as gene transfer vectors in the fields of transgenosis, gene capturing, gene therapy and the like, and have good application effects.
The early stage of this study found that the insertion sequence ISRP10 blocked the Pseudomonas aeruginosa oprD gene, resulting in resistance of the bacteria to imipenem (Qinghui Sun, et al, insertion sequence ISRP10 inactivation ofthe oprD gene inimipenem-resistant Pseudomonas aeruginosa clinical isolates [ J ]. International Journal of Antimibealalacents.2016, 5 (47): 375-379.) and that the positive rate of the insertion sequence was found to be 32.8% by detection of its positive rate. By analysis that ISRP10 belongs to IS30 family, the length IS 1115-1117bp, and the G+C content IS 60.22%. Amino acids 174-327 in the insert sequence ISRP10 code for an integrase having nuclease activity, exonuclease activity, hydrolase activity, DNA recombination function, DNA transfer function. There are also two metal active center sites in the catalytic domain, 184aa and 241aa, respectively, and both are magnesium ion active centers, as shown in FIG. 1.
Pseudomonas aeruginosa is a clinically important pathogenic bacterium, and not only can show natural drug resistance to various antibacterial drugs, but also can obtain drug resistance through antibiotic induction, and the drug resistance is more serious along with the wide application of the antibacterial drugs, so that the clinical treatment of the infection of the Pseudomonas aeruginosa becomes very difficult. The mechanism of generating drug resistance by pseudomonas aeruginosa is very complex, and the drug resistance mechanism can be studied by utilizing a transposon at present, but the current transposon library system is generally constructed by adopting a joint 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 transposon to be widely used, the invention provides a novel construction system of transposon mutation library, plasmids can be transferred into target strains through chemical transformation and electric transformation methods, and then expression of transposase is induced, and the transposon mutation library almost covering the whole genome range can be obtained by only one mutant strain containing transposon. The construction system of the novel transposon library not only can increase the diversity of transposition mutations, but also is beneficial to determining the insertion position of strains and researching genome annotation, functional analysis and drug resistance mechanism of pseudomonas aeruginosa.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems existing in the prior art and provides a novel construction system of a transposon mutant strain library, a novel transposon mutant library and application.
The technical scheme adopted for solving the technical problems is as follows:
a novel transposon mutant strain library construction system comprises the following construction steps:
(1) Simulating a natural transposon plasmid, and 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 transposon region, and the transposon region comprises transposase ISRP10 regulated by Plac, inverted terminal repeat ITRs and gentamycin resistance genes;
(2) Adding a transposase gene RP10 capable of being regulated by a Lac promoter gene into the novel transposon region;
(3) The recombinant plasmid is directly electrically transformed and guided into a receptor cell, and then the transposase expression is induced by IPTG to carry out transposition jump;
(4) The transposon mutant has the possibility of containing a novel transposon region at any position of the genome, the transposon region comprising: transposase ISRP10, terminal repeat ITRs and gentamycin resistance genes regulated by Plac;
(5) Amplifying and culturing a transposon mutant strain, wherein the number of cells reaches 10 11 At cfu/mL, IPTG with a final concentration of 1mM is added to induce the expression of the transposase, and the transposon is induced to jump for 2 hours, so that a transposon mutant library with a plurality of insertion gene forms within the whole genome is randomly obtained.
Further, the construction steps are specifically as follows:
(1) Simulating a natural transposon plasmid, and artificially constructing a novel transposon plasmid vector by replacing LacZ alpha gene in the pUCP20 plasmid with the novel transposon region on the basis of the pUCP20 transposon plasmid;
(2) The transposase RP10 of the novel transposon region can be regulated and controlled by the Lac promoter gene;
(3) Firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK by an electrochemical conversion method to obtain transposon mutant strains of different species containing transposons;
(4) Amplifying and culturing a transposon mutant strain, wherein the number of cells reaches 10 11 At cfu/mL, IPTG with a final concentration of 1mM was added, and the culture was induced for 2 hours to jump the transposon, thereby randomly obtaining a library of transposon mutants in the form of a plurality of inserted genes within the whole genome.
Further, the nucleotide sequence of the pUCP20 transposon plasmid is SEQ ID No.1.
Further, the pUCP20 transposon plasmid nucleotide sequence with LacZ alpha gene removed in the 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 transposon region in the step (A) is SEQ ID No.4.
Further, the nucleotide sequence of the novel transposon plasmid vector in the step A is SEQ ID No.5.
Furthermore, the concentration and volume of the transposon mutant strain can be controlled according to the requirements of library construction in the step, and the transposon jumping induction process of the transposase can be regulated and controlled.
A novel library of transposon mutations obtained using the construction system described above.
Use of the construction system described above in the construction of a library of pseudomonas aeruginosa insertion mutants.
The beneficial effects obtained by the invention are as follows:
1. construction of transposon mutant libraries is now widely used in genome functional annotation and functional analysis. At present, a transposon library system is generally constructed by adopting a joint transfer mode, but the efficiency of the transposon library constructed by the method is low and can only reach 10 -3 -10 -4 Moreover, the library obtained has a small capacity and cannot cover the whole genome of the bacteria. The invention constructs a novel transposon mutation library system by utilizing the advantage that pUCP20 plasmid has a wide host replication initiation region. The inserted sequence ISRP10 is firstly modified and cloned into pUCP20 plasmid to make it express and can be regulated by IPTG, and gentamicin resistance gene is added to aid screening. Obtaining a mutant strain containing transposon through electrotransformation, taking the mutant strain as an original strain, inducing the expression of novel transposase RP10 under the induction of IPTG, and jumping and inserting the transposon on different genes to obtain the transposon with rich varieties and multiple insertion gene formsA transposon mutation library which can be obtained by using only one mutant strain containing a transposon without requiring the efficiency of junction transfer. The construction system of the novel transposon library not only can increase the diversity of the transposition mutation, but also is helpful for determining the insertion position of the strain.
2. The construction system can systematically and efficiently obtain the pseudomonas aeruginosa transposon mutant strain library, and is helpful for researching the pseudomonas aeruginosa drug resistance mechanism.
Drawings
FIG. 1 is an integrase assay of ISRP10 in the present invention;
FIG. 2 is a genetic map of vector plasmid pUCP2021 according to the present invention.
Detailed Description
The following describes the embodiments of the present invention in detail, but the present embodiments are illustrative and not limitative, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A novel transposon mutant strain library construction system comprises the following construction steps:
(1) Mimicking 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 transposon region, and the transposon region comprises a transposase ISRP10 regulated by Plac, an inverted terminal repeat ITRs (invertedterminal repeats, ITRs) and a gentamycin resistance gene;
(2) Adding a transposase gene RP10 capable of being regulated by a Lac promoter gene into the novel transposon region;
(3) The recombinant plasmid is directly electrically transformed and guided into a receptor cell, and then the transposase expression is induced by IPTG to carry out transposition jump;
(4) The transposon mutant has the possibility of containing a novel transposon region at any position of the genome, the transposon region comprising: plac-regulated transposase ISRP10, terminal repeats ITRs (inverted terminal repeats, ITRs), and gentamicin resistance genes;
(5) Amplifying and culturing a transposon mutant strain, wherein the number of cells reaches 10 11 At cfu/mL, IPTG with a final concentration of 1mM is added to induce the expression of the transposase, and the transposon is induced to jump for 2 hours, so that a transposon mutant library with a plurality of insertion gene forms within the whole genome is randomly obtained.
Preferably, the construction steps are as follows:
(1) Simulating a natural transposon plasmid, and artificially constructing a novel transposon plasmid vector by replacing LacZ alpha gene in pUCP20 plasmid with a novel transposon region on the basis of pUCP20 transposon plasmid, wherein the genetic map is shown in figure 2;
(2) The transposase RP10 of the novel transposon region can be regulated and controlled by the Lac promoter gene;
(3) Firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK by an electrochemical conversion method to obtain transposon mutant strains of different species containing transposons; 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, wherein the number of cells reaches 10 11 At cfu/mL, IPTG with a final concentration of 1mM was added, and the culture was induced for 2 hours to jump the transposon, thereby randomly obtaining a library of transposon mutants in the form of a plurality of inserted genes within the whole genome.
Preferably, the pUCP20 transposon plasmid has the nucleotide sequence of SEQ ID No.1.
The transposon plasmid vector has the following characteristics:
(1) the pUCP20 transposon plasmid gene sequence from which the LacZ alpha gene region was deleted;
(2) adding a pre-constructed novel transposon region into the original LacZ alpha gene region, wherein the transposon region comprises: the Plac-regulated insert ISRP10, terminal repeats (inverted terminal repeats, ITRs) and gentamicin resistance genes;
preferably, the pUCP20 transposon plasmid with LacZ alpha gene removed in the step (A) has a nucleotide sequence of 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 transposon region in the step (A) is SEQ ID No.4.
Preferably, the nucleotide sequence of the novel transposon plasmid vector in the step (A) is SEQ ID No.5.
Preferably, the concentration and volume of the transposon mutant strain can be controlled according to the requirements of library construction in the step, and the transposon jump induction process of the transposase can be regulated.
A novel library of transposon mutations obtained using the construction system described above.
Use of the construction system described above in the construction of a library of pseudomonas aeruginosa insertion mutants.
Specifically, the related preparation and detection are as follows:
the vector of the invention takes pUCP20 plasmid preserved in a laboratory as a starting plasmid, firstly removes LacZ alpha gene of the pUCP20 plasmid, and adds a novel transposon region in the region, wherein the transposon region comprises: the Plac-regulated insert ISRP10, terminal repeats (inverted terminal repeats, ITRs) and the gentamicin resistance gene. Under the induction of IPTG, the transposon can jump to a new recipient bacterium genome together with the Plac-regulated insertion sequence ISRP10 gene and Gm resistance gene, and the transformed transposition plasmid is named pUCP2021. Electrochemically transferring pUCP2021 plasmid into PAK to obtain donor strain containing transposase gene RP10 and Gm resistance gene regulated by IPTG; when the recipient bacterium containing the transposon is cultured alone, the recipient bacterium in the form of a plurality of inserted genes can be obtained by the method of induction by adding IPTG.
The construction process of the transposon plasmid of the present invention and the construction process of the novel transposon library are described in detail below.
1. Construction of transposon plasmid (pUCP 2021)
First, designing primer amplification to obtain insertion sequence ISRP10 (in which the coding gene sequence of insertion sequence ISRP10 is SEQ ID NO. 3)
The clinical Pseudomonas aeruginosa TJ173 (the oprD gene of the strain has an insertion sequence ISRP 10) is used as a template, and an upstream primer is used: 5'-ATCTCGCAGGTTGGGTATGTTCATGCT-3' and downstream primers: 5'-CCACCATTCAAATCGGTCGTACCCAAG-3' the insertion sequence ISRP10 gene was obtained by amplification.
In the second step, the Gm resistance encoding gene sequence was derived from the laboratory preservation plasmid pEX18Gm.
Third, the laboratory preservation plasmid pUCP20 was deleted for LacZ alpha gene (nucleotide sequence SEQ ID NO. 2).
Fourth, adding Gm resistance of pEX18Gm plasmid at one end of ITRs of insertion sequence ISRP10 coding gene obtained by PCR, controlling expression of novel transposase RP10 by using Plac promoter of pUCP20, reconstructing novel transposon region together with pUCP20 skeleton with LacZ alpha gene removed to obtain novel transposon plasmid vector pUCP2021 (nucleotide sequence of SEQ ID No. 5)
This step was submitted to the Suzhou gold intelligent sequencing company in China to complete the 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 jump transposons to obtain a transposon library with a plurality of types and a large quantity, which covers the whole gene range.
2. Novel transposon library construction system
In the first step, the constructed plasmid pUCP2021 is electrically transferred into the target strain PAK, and the conversion solution is coated on the Amp 100 、Gm 100 On LB medium of (C), overnight culture to give a recipient strain containing the plasmid.
Secondly, extracting plasmids to verify whether the recipient bacteria contain target plasmids, and if the target plasmids are verified to be correct, the bacteria can be used as starting strains in the next step.
Third, the plasmid-containing strain was streaked and purified three times, and cultured overnight, at which time the antibody was raisedThe concentration of biotin is Amp 100 And Gm 100 。
Fourth, the overnight cultured strain was inoculated at 3% of the amount of the inoculated strain and cultured to OD 600 At=0.6 IPTG was added to a final concentration of 1mM to induce transposase expression, resulting in a genome-wide transposon library with multiple gene insertions.
Specifically:
1. the constructed pUCP2021 plasmid was transformed electrically into the recipient Pseudomonas aeruginosa PAK.
2. Coating the conversion solution on the Amp 100 And Gm 100 Is cultured overnight at 37℃for 12 hours to give a recipient strain containing the plasmid.
4. 10 transformants were selected for plasmid verification, and only strain No.3 of 10 strains was negative.
5. SQ1 was inoculated overnight at 3% of the transfer amount to give an OD by taking SQ1 as an initial strain of 10 strains 600 At=0.6 IPTG at a final concentration of 1mM was added to induce transposase expression, and the culture was induced for 2h, the transposase expression allowing the transposon to jump to other positions of the PAK strain genome, resulting in a whole genome transposon library covering multiple gene insertion forms of whole genes.
Seq1
Nucleotide sequence of pUCP20 transposon plasmid
Seq2
Nucleotide sequence of pUCP20 transposon plasmid from which LacZ alpha gene was deleted
Seq3.
Nucleotide sequence of insertion sequence ISRP10 coding gene
Seq4.
Nucleotide sequence of novel transposon region (RP 10) encoding gene
Seq5.
Nucleotide sequence of novel transposon plasmid vector
Although 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 disclosure of the embodiments.
Sequence listing
<110> university of Tianjin science and technology, hainan medical college
<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> nucleotide sequence of the pucp20 transposon plasmid from which laczα gene was deleted (Unknown)
<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 insertion sequence isrp10 coding gene (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 transposon region (nucleotide sequence Unknown of rp10 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 (5)
1. A transposon system for constructing a library of mutants, characterized in that: the construction steps are as follows:
(1) Imitate the natural transposon plasmid, artificially construct a pUCP20 transposon plasmid based on pUCP20 transposon plasmid by replacing the transposon regionLacZαTransposon plasmid vector of gene, transposon region including transposase IS under Plac controlRP10Reverse terminal repeats ITRs and gentamicin resistance genes;
(2) The transposase ISRP10 of the transposon region is capable ofLacRegulation of a promoter gene;
(3) Firstly, transferring the constructed transposon plasmid into pseudomonas aeruginosa PAK by an electrochemical conversion method to obtain a transposon mutant strain containing transposons;
(4) Amplifying and culturing a transposon mutant strain, wherein the number of cells reaches 10 11 At cfu/mL, adding IPTG with the final concentration of 1mM, inducing 2h to jump transposons, and randomly obtaining a transposon mutant strain library in a plurality of insertion gene forms within the whole genome range;
the nucleotide sequence of the transposon plasmid vector is SEQ ID No.5.
2. The transposon system of claim 1 wherein the transposon system is used to construct a library of mutants: the nucleotide sequence of the pUCP20 transposon plasmid is SEQ ID No.1.
3. The transposon system of claim 1 wherein the transposon system is used to construct a library of mutants: the transposase ISRP10The nucleotide sequence of (2) is SEQ ID NO.3.
4. The transposon system of claim 1 wherein the transposon system is used to construct a library of mutants: the nucleotide sequence of the transposon region in the step (A) is SEQ ID No.4.
5. Use of the system according to any one of claims 1 to 4 for the construction of a library of pseudomonas aeruginosa insertion mutants.
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