CN112626064B - Construction system of novel transposon mutant strain library - Google Patents
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Abstract
The invention relates to a construction system of a novel transposon mutant strain library, which comprises the following construction steps: constructing a novel transposon plasmid vector additionally added with a transposase gene tnpA in an ITRs region; secondly, the transposase gene tnpA additionally added in the ITRs region can be regulated and controlled by an arabinose promoter gene araBAD and an arabinose arrestin gene araC; obtaining a transposon mutant strain of different species containing the transposon; the transposon mutant has a transposon insertion sequence at any position of the genome; fifthly, randomly obtaining a transposon mutant strain library in the form of multiple inserted genes in the whole genome range. The construction system of the invention can systematically and efficiently obtain the transposon mutant library of gram-negative bacteria covering the whole genome, and is beneficial to the research of genome annotation and functional analysis.
Description
Technical Field
The invention belongs to the technical field of gene mutation, and particularly relates to a novel construction system of a transposon mutant strain library.
Background
With the rapid development of high-throughput sequencing technology, more and more biological genome annotation results show that: transposons are present in the genome of almost all organisms and are an important component of the genome of most organisms. Among them, tc1/Mariner transposons are a DNA transposon superfamily which is most widely distributed in nature, and 14 active Tc1/Mariner transposons (such as Minos, mos1 and the like) have been found in nature, and high activity artificial transposons such as Sleeping Beauty transposons (SB) are obtained by molecular reconstruction. Transposons such as SB and Mos1 have been widely used as gene transfer vectors in the research of the fields of transgenosis, gene capture, gene therapy and the like, and have achieved good application effects.
The Tc1/Mariner transposon family members typically have a full length of 1300-2400 bp and contain a single gene encoding transposase flanked by Inverted Terminal Repeats (ITRs). When transposases bind to ITRs, transposition can be promoted. Tc1/Mariner is an important genetic engineering tool that can be used to construct transposon systems by exploiting their transposable sequences and the ability of transposases to segregate. The current transposome 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 to make transposon use more widely, the invention provides a novel construction system of transposon mutation library, which does not require the efficiency of conjugal transfer and only needs one transposon-containing mutant strain to obtain the transposon mutation library almost covering the whole genome. The construction system of the novel transposon library can not only increase the diversity of transposition mutations, but also be helpful for determining the insertion position of the strain.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a construction system of a novel transposon mutant strain library and the transposon mutant library.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a construction system of a novel transposon mutant strain library comprises the following construction steps:
a novel transposon plasmid vector which additionally adds a transposase gene tnpA in an ITRs region is artificially constructed to mimic a natural transposon plasmid;
secondly, the transposase gene tnpA additionally added in the ITRs region is regulated and controlled by an arabinose promoter gene araBAD and an arabinose arrestin gene araC;
thirdly, pelleting the transposon substances to be transferred into E.coli S17-1 lambda pir, and then obtaining a transposon mutant strain of different species containing the transposon by utilizing a common conjugal transfer method;
the transposon mutant has a transposon insertion sequence at any position of the genome, and the insertion sequence comprises: ITRs regions at two ends, an arabinose promoter gene araBAD, an arabinose arrestin gene araC and a transposase gene tnpA;
fifthly, amplifying and culturing a transposon mutant strain until the number of cells reaches 10 12 At cfu/mL, arabinose was added to the final concentration of 2mg/mL, and induction culture was carried out for 2 hours to hop the transposon, thereby randomly obtaining a transposon mutant library of various inserted gene forms in the whole genome.
Moreover, the transposon plasmid vector of the step.
(1) The pKKma transposon plasmid gene sequence from which the tnpA transposase gene was deleted;
(2) an arabinose promoter gene araBAD and an arabinose arrestin gene araC are added in the ITRs region;
(3) the transposase gene tnpA regulated by arabinose was added to the region of ITRs.
Moreover, the nucleotide sequence of the pKKma transposon plasmid is SEQ ID NO.1.
Moreover, the nucleotide sequence of the gene of the tnpA transposase in the first step is SEQ ID No.3.
Moreover, the nucleotide sequence of the novel transposon plasmid vector in the step is SEQ ID NO.4.
And in the step three, the transposon plasmid vector is transformed into E.coli S17-1 lambda pir to obtain an escherichia coli strain containing the transposase gene tnpA which is stably expressed and regulated by arabinose, and then a common conjugal transfer mating method is utilized to obtain a transposon mutant strain of other species containing the transposon.
And the 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.
And the nucleotide sequences of the coding arabinose promoter gene araBAD and the arabinose arrestin gene araC in the step II are SEQ ID NO.2.
A novel transposon mutation library obtained by using the construction system of the novel transposon mutant library as described above.
The invention has the advantages and positive effects that:
1. construction of transposon mutant libraries is now widely used for functional annotation and functional analysis of genomes. 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 the transposition efficiency can only reach 10 -3 -10 -4 Moreover, the resulting library is of a small capacity and cannot cover the entire genome of the bacteria. The invention provides a construction system of a novel transposon mutation library, transposase gene tnpA is firstly cloned to an Inverted Terminal Repeat (ITRs) region, the expression of the transposition gene tnpA is regulated and controlled by an arabinose promoter gene araBAD and an arabinose arrestin gene araC, a 'two-step method' is adopted, firstly, a mutant strain containing transposon is obtained through conjugal transfer, the mutant strain is taken as a starting strain, the ITRs are jumped and inserted into different genes under the induction of arabinose, and the transposon mutation library with various insertion gene forms is obtained in rich varieties. 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 of the invention can systematically and efficiently obtain the transposon mutant library of the gram-negative bacteria covering the whole genome, and is beneficial to researching the genome annotation and functional analysis of the gram-negative bacteria.
Drawings
FIG. 1 is a genetic map of the vector plasmid pLDH2001 in the present invention; wherein KpnI and PstI are two enzyme cutting sites for connecting pKKma delta tnPA plasmid and purified mariner transposase gene tnPA regulated by arabinose.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
A construction system of a novel transposon mutant strain library comprises the following construction steps:
first, a novel transposon plasmid vector in which a transposase gene tnpA was additionally added to an ITRs region was artificially constructed in a manner similar to a natural transposon plasmid, and its genetic map is shown in fig. 1. (ii) a
Secondly, the transposase gene tnpA additionally added in the ITRs region can be regulated and controlled by an arabinose promoter gene araBAD and an arabinose arrestin gene araC;
thirdly, pelleting the transposon substances to be transferred into E.coli S17-1 lambda pir, and then obtaining a transposon mutant strain of different species containing the transposon by utilizing a common conjugal transfer method;
the transposon mutant has a transposon insertion sequence at any position of the genome, and the insertion sequence comprises: ITRs regions at two ends, an arabinose promoter gene araBAD, an arabinose arrestin gene araC and a transposase gene tnpA;
fifthly, amplifying and culturing a transposon mutant strain until the number of cells reaches 10 12 At cfu/mL, arabinose was added to the final concentration of 2mg/mL, and induction culture was carried out for 2 hours to hop the transposon, thereby randomly obtaining a transposon mutant library of various inserted gene forms in the whole genome.
Preferably, the transposon plasmid vector of the step.
(1) The pKKma transposon plasmid gene sequence from which the tnpA transposase gene was deleted;
(2) an arabinose promoter gene araBAD and an arabinose arrestin gene araC are added in the ITRs region;
(3) the transposase gene tnpA regulated by arabinose was added to the region of ITRs.
Preferably, the nucleotide sequence of the pKKma transposon plasmid is SEQ ID NO.1.
Preferably, the nucleotide sequence of the gene of tnpA transposase in the first step is SEQ ID No.3.
Preferably, the nucleotide sequence of the novel transposon plasmid vector of the step is SEQ ID No.4.
Preferably, in the step three, the transposon plasmid vector is transformed into E.coli S17-1 lambda pir to obtain an escherichia coli strain containing the transposase gene tnpA which is stably expressed and regulated by arabinose, and then a common conjugal transfer mating method is utilized to obtain a transposon mutant strain of other species containing the transposon.
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.
Preferably, the nucleotide sequences of the arabinose promoter gene araBAD and the arabinose arrestin gene araC in the step II are SEQ ID NO.2.
A novel transposon mutation library obtained by using the construction system of the novel transposon mutant library as described above.
Specifically, the preparation and detection are as follows:
the vector takes a pKKma transposon plasmid which is preserved in a laboratory and has a mariner transposase gene as a starting plasmid, removes tnpA transposase of the pKKma plasmid, adds a tnpA transposase gene regulated by arabinose into an ITRs region, can jump to a new receptor bacterium genome together with the transposase gene induced by the arabinose under the induction of the arabinose, and names the transformed transposable plasmid pLDH2001. Plasmatizing pLDH2001 into E.coli S17-1 lambda pir to obtain a transposition donor strain containing a stably expressed and arabinose-regulated transposase gene tnpA; and (3) carrying out mating on the donor bacteria and different recipient bacteria to obtain the recipient bacteria containing the transposon, wherein the recipient bacteria have gentamicin resistance and the transposase gene tnpA is regulated and controlled by arabinose. When a recipient strain containing a transposon is cultured alone, a plurality of recipient strains in the form of inserted genes can be obtained by an arabinose-induced method.
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.
1. Construction of transposon plasmid (pLDH 2001)
First, primers were designed to remove the mariner transposase gene tnpA of the mariner transposon plasmid pKKma plasmid (Zhang Cuikun, dan Litao, and Yang Hongjiang. Sequence analysis and transposition performance of the mariner transposon pKKma [ J ]. Microbiology report, 2015,55 (03): 366-371, a laboratory-stored plasmid, genBank sequence accession number KJ 933396) (where the sequence of transposase gene tnpA is SEQ ID NO: 3).
pKKma plasmid was used as template, with upstream primer: 5'-GATCCTCCAATTCGCCCTATAG-3' and downstream primer: 5363 and amplifying a pKKma delta tnpA fragment (wherein the sequence of the pKKma vector with the tnpA transposase removed is SEQ ID NO: 1) by 5'-CTAATACTAGCGACGCCATCTATGT-3', purifying and recovering the target fragment by a gel cutting mode, phosphorylating the recovered fragment, recovering the target fragment from liquid, diluting the recovered fragment by 10 times in a gradient manner, uniformly mixing the diluted fragment with Solution I in the same volume, connecting the mixture at 16 ℃ overnight to obtain a pKKma delta tnpA plasmid, granulating the connected pKKma delta tnpA plasmid into escherichia coli DH5 alpha, culturing the obtained product overnight, and carrying out enzyme digestion to verify the size of the plasmid to obtain the verified correct pKKma delta tnpA plasmid.
Secondly, a primer is designed and amplified, and the arabinose promoter gene araBAD and the arabinose repressor gene araC (wherein the sequences of the arabinose promoter gene araBAD and the arabinose repressor gene araC are SEQ ID NO: 2) on the plasmid pCasPA (Weizhong, chen, ya, et. CRISPR/Cas9-based Genome amplification in Pseudomonas aeruginosa and cytosine deamidase-media Base amplification in Pseudomonas specificities [ J ]. Isscience, 2018.) are stored in a laboratory.
Using pCasPA plasmid as a template, and using an upstream primer: 5' -CGGGGTACCTTCCTGCGTTGTCGACTGTA-3' and the downstream primer: 5' -CGAAATTCCTTTTTTTCCATTTTTTATAACCTCCTTAGAGCTCGA-3' amplifying arabinose promoter gene araBAD and arabinose arrestin gene araC, aiming at using the arabinose promoter gene araBAD and the arabinose arrestin gene araC to regulate the expression of mariner transposase tnpA, in order to connect with the mariner transposase tnpA and a target vector pKKma delta tnpA plasmid, so a KpnI enzyme cutting site and a 20bp homologous sequence with the mariner transposase tnpA gene are added during the design of a primer, and a target fragment is purified and recovered by a gel cutting mode.
Thirdly, designing primers to amplify mariner transposase gene tnpA of pKKma plasmid
Taking pKKma plasmid as a template, and utilizing an upstream primer: 5'-TTGTGAAGCAACTTTTGTTATTGTG-3' and downstream primer: 5' -AACTGCAG5363 and amplifying mariner transposase gene tnpA by GGAGGATCCGGTCTAACAAAG-3' to connect with a target vector pKKma delta tnpA plasmid, so that a PstI enzyme cutting site is added when a primer is designed, and a target fragment is purified and recovered by a gel cutting mode.
Fourthly, the pKKma delta tnpA, the arabinose promoter gene araBAD, the arabinose repressor gene araC and the mariner transposase gene tnPA are connected into a complete plasmid by utilizing homologous recombination and enzyme digestion recombination, and the complete plasmid is named as pLDH2001 (the sequence of the pLDH2001 is SEQ ID NO: 4).
Connecting the purified and recovered fragment arabinose promoter gene araBAD with the arabinose arrestin gene araC and mariner transposase gene tnpA by using homologous recombinase in a water bath at 37 ℃ for 30min, and then using an upstream primer: 5' -CGGGGTACCTTCCTGCGTTGTCGACTGTA-3' and the downstream primer: 5' -AACTGCAGGGAGGATCCGGTCTAACAAAG-3' amplifying to obtain connected mariner transposase gene tnpA induced by arabinose, purifying and recovering by means of gel cuttingA fragment of interest. Carrying out double enzyme digestion on pKKMMa delta tnPA plasmid and purified mariner transposase gene tnPA induced by arabinose by KpnI and PstI, carrying out gel cutting, purifying and recovering a target fragment after enzyme digestion, mixing the recovered fragment and Solution I in equal volume, connecting overnight at 16 ℃ to obtain pLDH2001 plasmid, granulating and transferring the connected pLDH2001 plasmid into escherichia coli DH5 alpha, carrying out overnight culture, and carrying out enzyme digestion to verify the size of the plasmid to obtain the correct pLDH2001 plasmid.
And fifthly, successfully transforming the plasmid pLDH2001 into a strain E.coli S17-1 lambda pir to obtain a recombinant strain E.coli S17-1 lambda pir/pLDH2001, namely a novel transposition system strain, wherein the strain can generate transposase through arabinose induction to enable transposons to jump to obtain a transposable library which covers the whole gene range and has multiple types and large quantity.
2. Novel construction system of transposable library
First, culture donor bacterium E.coli S17-1 lambda pir/pLDH2001 until the bacterial concentration reaches 10 8 cfu/mL, mixing donor bacteria and other recipient bacteria by means of conjugal transfer, culturing the mixed bacteria on a beef extract culture medium containing arabinose with the final concentration of 2mg/mL for 12h, inducing transposase expression by using the arabinose to enable transposons to jump to the recipient strains, sucking overnight bacterial sludge by blowing and uniformly mixing the bacterial sludge with liquid LB, and coating the bacterial sludge on Gm in a gradient dilution manner 80 The recipient strain containing the transposon was grown overnight on the resistant plates.
And secondly, verifying whether the strain on the colony plate contains an arabinose promoter gene araBAD, an arabinose arrestin gene araC and a mariner transposase gene tnpA by PCR, and if the PCR result is positive, obtaining the strain which is successfully subjected to conjugation transfer and contains a resistance gene Gm and a transposase gene tnpA controlled by arabinose, wherein the strain can be used as a starting strain in the next step.
Thirdly, the transposon-containing strain was streaked and purified three times and cultured overnight.
The fourth step, the overnight cultured strain was transferred at 3% transfer amount and cultured to OD 600 Adding arabinose with final concentration of 2mg/ml when the concentration is 0.6, inducing transposase expression, and obtaining the complete gene insertedA library of genomic transposomes.
Specifically, the method comprises the following steps:
1. 3% of transfer amount is used for transferring donor bacteria E. Coli S17-1 lambda pir/pLDH2001 which are cultured overnight to a final concentration of Gm 3 300mL of LB medium, and cultured at 37 ℃ for 10 hours. The overnight cultured recipient bacterium Pseudomonas fluorescens X was inoculated into 100mL of LB medium at 3% of the inoculum size, and cultured at 30 ℃ for 10 hours.
2. The donor and recipient were centrifuged at 6000rpm for 10min, and the sludge pellet was mixed well and cultured on a beef extract medium containing arabinose at a final concentration of 2mg/ml for 12 hours.
3. The overnight bacterial sludge is evenly blown and sucked by liquid LB, diluted by 10 times and coated on Gm in a gradient way 80 The transposon-containing recipient strain was obtained by culturing the plates overnight at 30 ℃ for 12 hours.
4. 10 receptor strains are selected for streak purification, 2 times of purification are carried out, PCR verifies whether the 10 receptor strains contain an arabinose promoter gene araBAD, an arabinose arrestin gene araC and a mariner transposase gene tnpA, and only the No.4 strain of the 10 receptor strains is a negative result.
5. Transferring one strain QM1 of 10 strains as an initial strain to the overnight-cultured QM1 with the transfer amount of 3 percent, and culturing to OD 600 And when the final concentration of arabinose is 2mg/ml when the total gene is 0.6, induction culture is carried out for 2h, and transposase expression enables transposons to jump to other positions of the genome of the QM1 strain, so that a genome-wide transposon library covering various gene insertion forms of the whole gene is obtained.
Seq1
pKKma plasmid from which transposase gene tnpA was deleted
Seq2
Arabinose promoter gene araBAD and arabinose repressor gene araC
Seq3.
Transposase gene tnpA
Seq4.
pLDH2001
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> Tianjin science and technology university
<120> construction system of novel transposon mutant library
<160> 12
<170> SIPOSequenceListing 1.0
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<211> 5368
<212> DNA/RNA
<213> pKKma plasmid (Unknown) from which transposase gene tnpA was deleted
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gctttctgtc ctagagaccg gggacttatc agccaacctg ttactcccgg gacccagctt 60
tcttgtacag ggcacaattc ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg 120
ctttgagagg ctctaagggc ttctcagtgc gttacatccc tggcttgttg tccacaaccg 180
ttaaacctta aaagctttaa aagccttata tattcttttt tttcttataa aacttaaaac 240
cttagaggct atttaagttg ctgatttata ttaattttat tgttcaaaca tgagagctta 300
gtacgtgaaa catgagagct tagtacgtta gccatgagag cttagtacgt tagccatgag 360
ggtttagttc gttaaacatg agagcttagt acgttaaaca tgagagctta gtacgtgaaa 420
catgagagct tagtacgtac tatcaacagg ttgaactgct gatcttcaga tcctctacgc 480
cggacgcatc gtggccggat ccagccgacc aggctttcca cgcccgcgtg ccgctccatg 540
tcgttcgcgc ggttctcgga aacgcgctgc cgcgtttcgt gattgtcacg ctcaagcccg 600
tagtcccgtt cgagcgtcgc gcagaggtca gcgagagcgc ggtaggcccg atacggctca 660
tggatggtgt ttcgggtcgg gtgaatcttg ttgatggcga tatggatgtg caggttgtcg 720
gtgtcgtgat gcacggcact gacgcgctga tgctcggcga agccaagccc agcgcagatg 780
cggtcctcaa tcgcgcgcaa cgtctccgcg tcgggcttct ctcccgcgcg gaagctaacc 840
agcaggtgat aggtcttgtc ggcctcggaa cgggtgttgc cgtgctgggt cgccatcacc 900
tcggccatga cagcgggcag ggtgtttgcc tcgcagttcg tgacgcgcac gtgacccagg 960
cgctcggtct tgccttgctc gtcggtgatg tacttcacca gctccgcgaa gtcgctcttc 1020
ttgatggagc gcatggggac gtgcttggca atcacgcgca ccccccggcc gttttagcgg 1080
ctaaaaaagt catggctctg ccctcgggcg gaccacgccc atcatgacct tgccaagctc 1140
gtcctgcttc tcttcgatct tcgccagcag ggcgaggatc gtggcatcac cgaaccgcgc 1200
cgtgcgcggg tcgtcggtga gccagagttt cagcaggccg cccaggcggc ccaggtcgcc 1260
attgatgcgg gccagctcgc ggacgtgctc atagtccacg acgcccgtga ttctgtagcc 1320
ctggccgacg gccagcaggt aggccgacag gctcatgccg gccgccgccg ccttttcctc 1380
aatcgctctt cgttcgtctg gaaggcagta caccttgata ggtgggctgc ccttcctggt 1440
tggcttggtt tcatcagcca tccgcttgcc ctcatctgtt acgccggcgg tagccggcca 1500
gcctcgcaga gcaggattcc cgttgagcac cgccaggtgc gaataaggga cagtgaagaa 1560
ggaacacccg ctcgcgggtg ggcctacttc acctatcctg cccggctgac gccgttggat 1620
acaccaagga aagtctacac gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga 1680
tggatatacc gaaaaaatcg ctataatgac cccgaagcag ggttatgcag cggaaaagcg 1740
ctgcttccct gctgttttgt ggaatatcta ccgactggaa acaggcaaat gcgggaaatt 1800
actgaactga ggggacaggc gagagacgat gccaaagagc tacaccgacg agctggccga 1860
gtgggttgaa tcccgcgcgg ccaagaagcg ccggcgtgat gaggctgcgg ttgcgttcct 1920
ggcggtgagg gcggatgtcg aggcggcgtt agcgtccggc tatgcgctcg tcaccatttg 1980
ggagcacatg cgggaaacgg ggaaggtcaa gttctcctac gagacgttcc gctcgcacgc 2040
caggcggcac atcaaggcca agcccgccga tgtgcccgca ccgcaggcca aggctgcgga 2100
acccgcgccg gcacccaaga cgccggagcc acggcggccg aagcaggggg gcaaggctga 2160
aaagccggcc cccgctgcgg ccccgaccgg cttcaccttc aacccaacac cggacaaaaa 2220
ggatccacta gttctagagc ggccgctggc acttttcggg gaaatgtgcg cggaacccct 2280
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2340
taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2400
cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 2460
aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 2520
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 2580
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 2640
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 2700
catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 2760
aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 2820
ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 2880
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 2940
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3000
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3060
gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3120
gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3180
gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3240
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3300
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3360
ttccactgag cgtcagaccc cgtagagcgg ccgccagtgt gatggatgac acatagatgg 3420
cgtcgctagt attaggatcc tccaattcgc cctatagtga gtcgtattac gcgcgctcac 3480
tggccgtcgt tttacagcgt cgtgactggg aaaacccttg cgttacccaa cttaatcgcc 3540
ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accggtgccc 3600
tgtacaagaa agctgggtcc cgggagtaac aggttggctg ataagtcccc ggtctctaga 3660
aagcaggctt ttagacgcat cccgttccat acmsrmsccg ggcgaacaaa cgatgctcgc 3720
cttccagaaa accgaggatg cgaaccactt catccggggt cagcaccacc ggcaagcgcc 3780
gcgacggccc gcgtcggccg ggaagccgat ctcggcttga acgaattgtt aggtggcggt 3840
acttgggtcg atatcaaagt gcatcacttc ttcccgtatg cccaactttg tatagagagc 3900
cactgcggga tcgtcaccgt aatctgcttg cacgtagatc acataagcac caagcgcgtt 3960
ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg ccctgcctcc ggtgctcgcc 4020
ggagactgcg agatcataga tatagatctc actacgcggc tgctcaaact tgggcagaac 4080
gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag gcagcaagcg cgatgaatgt 4140
cttactacgg agcaagttcc cgaggtaatc ggagtccggc tgatgttggg agtaggtggc 4200
tacgtctccg aactcacgac cgaaaagatc aagagcagcc cgcatggatt tgacttggtc 4260
agggccgagc ctacatgtgc gaatgatgcc catacttgag ccacctaact ttgttttagg 4320
gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac atcgttgctg ctccataaca 4380
tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta 4440
caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt 4500
cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac agtttacgaa 4560
ccgaacaggc ttatgtcaat tcgagaattg acgcgcgcgc agatcagttg gaagaatttg 4620
tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acaattcgtt 4680
caagccgacg ccgcttcgcg gcgcggctta actcaagcgt tagatgcact aagcacataa 4740
ttgctcacag ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata 4800
agccctacac aaattgggag atataccatg aaaggctggc tttttcttgt tatcgcaata 4860
gttggcgaag taatcgcaac atccgcatta aaatctagcg agggctttac taagctgatc 4920
cggtggatga ccttttgaat gacctttaat agattatatt actaattaat tggggaccct 4980
agaggtcccc ttttttattt taaaaatttt ttcacaaaac ggtttacaag cataaagctt 5040
gcatgcctgc aggtcgacgg atccccggga atctctagaa ttctcatgtt tgacagctta 5100
tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta 5160
tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct 5220
ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt 5280
tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac 5340
aggaaacagg actctagagg atcaccca 5368
<210> 2
<211> 1190
<212> DNA/RNA
<213> arabinose promoter gene araBAD and arabinose repressor gene araC (Unknown)
<400> 2
atggagaaac agtagagagt tgcgataaaa agcgtcaggt aggatccgct aatcttatgg 60
ataaaaatgc tatggcatag caaagtgtga cgccgtgcaa ataatcaatg tggacttttc 120
tgccgtgatt atagacactt ttgttacgcg tttttgtcat ggctttggtc ccgctttgtt 180
acagaatgct tttaataagc ggggttaccg gtttggttag cgagaagagc cagtaaaaga 240
cgcagtgacg gcaatgtctg atgcaatatg gacaattggt ttcttctctg aatggcggga 300
gtatgaaaag tatggctgaa gcgcaaaatg atcccctgct gccgggatac tcgtttaatg 360
cccatctggt ggcgggttta acgccgattg aggccaacgg ttatctcgat ttttttatcg 420
accgaccgct gggaatgaaa ggttatattc tcaatctcac cattcgcggt cagggggtgg 480
tgaaaaatca gggacgagaa tttgtttgcc gaccgggtga tattttgctg ttcccgccag 540
gagagattca tcactacggt cgtcatccgg aggctcgcga atggtatcac cagtgggttt 600
actttcgtcc gcgcgcctac tggcatgaat ggcttaactg gccgtcaata tttgccaata 660
cggggttctt tcgcccggat gaagcgcacc agccgcattt cagcgacctg tttgggcaaa 720
tcattaacgc cgggcaaggg gaagggcgct attcggagct gctggcgata aatctgcttg 780
agcaattgtt actgcggcgc atggaagcga ttaacgagtc gctccatcca ccgatggata 840
atcgggtacg cgaggcttgt cagtacatca gcgatcacct ggcagacagc aattttgata 900
tcgccagcgt cgcacagcat gtttgcttgt cgccgtcgcg tctgtcacat cttttccgcc 960
agcagttagg gattagcgtc ttaagctggc gcgaggacca acgtatcagc caggcgaagc 1020
tgcttttgag caccacccgg atgcctatcg ccaccgtcgg tcgcaatgtt ggttttgacg 1080
atcaactcta cttctcgcgg gtatttaaaa aatgcaccgg ggccagcccg agcgagttcc 1140
gtgccggttg tgaagaaaaa gtgaatgatg tagccgtcaa gttgtcataa 1190
<210> 3
<211> 1047
<212> DNA/RNA
<213> transposase gene tnpA (Unknown)
<400> 3
ttattcaaca tagttccctt caagagcgat acaacgatta taacgacctt ccaatttttt 60
gataccattt tggtagtact ccttcggttt tgcctcaaaa taggcctcag tttcggcgat 120
cacctcttca ttgcagccaa attttttccc tgcgagcatc cttttgaggt ctgagaacaa 180
gaaaaagtcg ctgggggcca gatctggaga atacggcggg tggggaagca attcgaagcc 240
caattcatga atttttgcca tcgttctcaa tgacttgtgg cacggtgcgt tgtcttggtg 300
gaacaacact tttttcttct tcatgtgggg ccgttttgcc gcgatttcga ccttcaaacg 360
ctccaataac gccatataat agtcactgtt gatggttttt cccttctcaa gataatcgat 420
aaaaattatt ccatgcgcat cccaaaaaac agaggccatt actttgccag cggacttttg 480
agtctttcca cgcttcggag acggttcacc ggtcgctgtc cactcagccg actgtcgatt 540
ggactcagga gtgtagtgat ggagccatgt ttcatccatt gtcacatatc gacggaaaaa 600
ctcgggtgta ttacgagtta acagctgcaa acaccgctta gaatcatcaa cacgtcgttg 660
tttttggtca aatgtgagct cgcgcggcac ccatttcgca cagagcttcc gcatatccaa 720
atattgatga atgatatgac caacacgttc ctttgatatc tttaaggcct ctgctatctc 780
gatcaacttc attttacggt cattcaaaat cattttgtgg atttttttga tgttttcgtc 840
ggtaaccacc tctttcgggc gtccactgcg ttcaccgtcc tccgtgctca tttcaccacg 900
cttgaatttt gcataccaat caattattgt tgatttccct ggggcagagt ccggaaactc 960
attatcaagc caagtttttg cttccactgt attttttccc ttcagaaaac agtattttat 1020
caaaacacga aattcctttt tttccat 1047
<210> 4
<211> 7651
<212> DNA/RNA
<213> nucleotide sequence of novel transposon plasmid vector (Unknown)
<400> 4
cggggtaccc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt 60
caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt 120
gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc 180
tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa 240
ttgtgagcgg ataacaattt cacacaggaa acaggactct agaggatcac ccagctttct 300
gtcctagaga ccggggactt atcagccaac ctgttactcc cgggacccag ctttcttgta 360
cagggcacaa ttcccatgtc agccgttaag tgttcctgtg tcactcaaaa ttgctttgag 420
aggctctaag ggcttctcag tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc 480
ttaaaagctt taaaagcctt atatattctt ttttttctta taaaacttaa aaccttagag 540
gctatttaag ttgctgattt atattaattt tattgttcaa acatgagagc ttagtacgtg 600
aaacatgaga gcttagtacg ttagccatga gagcttagta cgttagccat gagggtttag 660
ttcgttaaac atgagagctt agtacgttaa acatgagagc ttagtacgtg aaacatgaga 720
gcttagtacg tactatcaac aggttgaact gctgatcttc agatcctcta cgccggacgc 780
atcgtggccg gatccagccg accaggcttt ccacgcccgc gtgccgctcc atgtcgttcg 840
cgcggttctc ggaaacgcgc tgccgcgttt cgtgattgtc acgctcaagc ccgtagtccc 900
gttcgagcgt cgcgcagagg tcagcgagag cgcggtaggc ccgatacggc tcatggatgg 960
tgtttcgggt cgggtgaatc ttgttgatgg cgatatggat gtgcaggttg tcggtgtcgt 1020
gatgcacggc actgacgcgc tgatgctcgg cgaagccaag cccagcgcag atgcggtcct 1080
caatcgcgcg caacgtctcc gcgtcgggct tctctcccgc gcggaagcta accagcaggt 1140
gataggtctt gtcggcctcg gaacgggtgt tgccgtgctg ggtcgccatc acctcggcca 1200
tgacagcggg cagggtgttt gcctcgcagt tcgtgacgcg cacgtgaccc aggcgctcgg 1260
tcttgccttg ctcgtcggtg atgtacttca ccagctccgc gaagtcgctc ttcttgatgg 1320
agcgcatggg gacgtgcttg gcaatcacgc gcaccccccg gccgttttag cggctaaaaa 1380
agtcatggct ctgccctcgg gcggaccacg cccatcatga ccttgccaag ctcgtcctgc 1440
ttctcttcga tcttcgccag cagggcgagg atcgtggcat caccgaaccg cgccgtgcgc 1500
gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc ggcccaggtc gccattgatg 1560
cgggccagct cgcggacgtg ctcatagtcc acgacgcccg tgattctgta gccctggccg 1620
acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 1680
cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 1740
gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 1800
agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 1860
ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 1920
ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 1980
accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 2040
cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcgggaa attactgaac 2100
tgaggggaca ggcgagagac gatgccaaag agctacaccg acgagctggc cgagtgggtt 2160
gaatcccgcg cggccaagaa gcgccggcgt gatgaggctg cggttgcgtt cctggcggtg 2220
agggcggatg tcgaggcggc gttagcgtcc ggctatgcgc tcgtcaccat ttgggagcac 2280
atgcgggaaa cggggaaggt caagttctcc tacgagacgt tccgctcgca cgccaggcgg 2340
cacatcaagg ccaagcccgc cgatgtgccc gcaccgcagg ccaaggctgc ggaacccgcg 2400
ccggcaccca agacgccgga gccacggcgg ccgaagcagg ggggcaaggc tgaaaagccg 2460
gcccccgctg cggccccgac cggcttcacc ttcaacccaa caccggacaa aaaggatcca 2520
ctagttctag agcggccgct ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 2580
tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 2640
ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 2700
ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 2760
aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 2820
gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 2880
ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 2940
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 3000
cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 3060
cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 3120
acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 3180
caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 3240
taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 3300
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 3360
aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 3420
agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 3480
atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 3540
tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 3600
tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 3660
gagcgtcaga ccccgtagag cggccgccag tgtgatggat gacacataga tggcgtcgct 3720
agtattagga tcctccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt 3780
cgttttacag cgtcgtgact gggaaaaccc ttgcgttacc caacttaatc gccttgcagc 3840
acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccggtg ccctgtacaa 3900
gaaagctggg tcccgggagt aacaggttgg ctgataagtc cccggtctct agaaagcagg 3960
cttttagacg catcccgttc catacmsrms ccgggcgaac aaacgatgct cgccttccag 4020
aaaaccgagg atgcgaacca cttcatccgg ggtcagcacc accggcaagc gccgcgacgg 4080
cccgcgtcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 4140
tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 4200
ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 4260
tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 4320
gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc 4380
gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 4440
cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 4500
ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 4560
agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 4620
ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 4680
tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 4740
acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 4800
ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 4860
ggcttatgtc aattcgagaa ttgacgcgcg cgcagatcag ttggaagaat ttgtccacta 4920
cgtgaaaggc gagatcacca aggtagtcgg caaataatgt ctaacaattc gttcaagccg 4980
acgccgcttc gcggcgcggc ttaactcaag cgttagatgc actaagcaca taattgctca 5040
cagccaaact atcaggtcaa gtctgctttt attattttta agcgtgcata ataagcccta 5100
cacaaattgg gagatatacc atgaaaggct ggctttttct tgttatcgca atagttggcg 5160
aagtaatcgc aacatccgca ttaaaatcta gcgagggctt tactaagctg atccggtgga 5220
tgaactgcag ggaggatccg gtctaacaaa gaaaaacaca tttttttgtg aaaattcgtt 5280
tttattattc aacatagttc ccttcaagag cgatacaacg attataacga ccttccaatt 5340
ttttgatacc attttggtag tactccttcg gttttgcctc aaaataggcc tcagtttcgg 5400
cgatcacctc ttcattgcag ccaaattttt tccctgcgag catccttttg aggtctgaga 5460
acaagaaaaa gtcgctgggg gccagatctg gagaatacgg cgggtgggga agcaattcga 5520
agcccaattc atgaattttt gccatcgttc tcaatgactt gtggcacggt gcgttgtctt 5580
ggtggaacaa cacttttttc ttcttcatgt ggggccgttt tgccgcgatt tcgaccttca 5640
aacgctccaa taacgccata taatagtcac tgttgatggt ttttcccttc tcaagataat 5700
cgataaaaat tattccatgc gcatcccaaa aaacagaggc cattactttg ccagcggact 5760
tttgagtctt tccacgcttc ggagacggtt caccggtcgc tgtccactca gccgactgtc 5820
gattggactc aggagtgtag tgatggagcc atgtttcatc cattgtcaca tatcgacgga 5880
aaaactcggg tgtattacga gttaacagct gcaaacaccg cttagaatca tcaacacgtc 5940
gttgtttttg gtcaaatgtg agctcgcgcg gcacccattt cgcacagagc ttccgcatat 6000
ccaaatattg atgaatgata tgaccaacac gttcctttga tatctttaag gcctctgcta 6060
tctcgatcaa cttcatttta cggtcattca aaatcatttt gtggattttt ttgatgtttt 6120
cgtcggtaac cacctctttc gggcgtccac tgcgttcacc gtcctccgtg ctcatttcac 6180
cacgcttgaa ttttgcatac caatcaatta ttgttgattt ccctggggca gagtccggaa 6240
actcattatc aagccaagtt tttgcttcca ctgtattttt tcccttcaga aaacagtatt 6300
ttatcaaaac acgaaattcc tttttttcca ttttttataa cctccttaga gctcgaattc 6360
ccaaaaaaac gggtatggag aaacagtaga gagttgcgat aaaaagcgtc aggtaggatc 6420
cgctaatctt atggataaaa atgctatggc atagcaaagt gtgacgccgt gcaaataatc 6480
aatgtggact tttctgccgt gattatagac acttttgtta cgcgtttttg tcatggcttt 6540
ggtcccgctt tgttacagaa tgcttttaat aagcggggtt accggtttgg ttagcgagaa 6600
gagccagtaa aagacgcagt gacggcaatg tctgatgcaa tatggacaat tggtttcttc 6660
tctgaatggc gggagtatga aaagtatggc tgaagcgcaa aatgatcccc tgctgccggg 6720
atactcgttt aatgcccatc tggtggcggg tttaacgccg attgaggcca acggttatct 6780
cgattttttt atcgaccgac cgctgggaat gaaaggttat attctcaatc tcaccattcg 6840
cggtcagggg gtggtgaaaa atcagggacg agaatttgtt tgccgaccgg gtgatatttt 6900
gctgttcccg ccaggagaga ttcatcacta cggtcgtcat ccggaggctc gcgaatggta 6960
tcaccagtgg gtttactttc gtccgcgcgc ctactggcat gaatggctta actggccgtc 7020
aatatttgcc aatacggggt tctttcgccc ggatgaagcg caccagccgc atttcagcga 7080
cctgtttggg caaatcatta acgccgggca aggggaaggg cgctattcgg agctgctggc 7140
gataaatctg cttgagcaat tgttactgcg gcgcatggaa gcgattaacg agtcgctcca 7200
tccaccgatg gataatcggg tacgcgaggc ttgtcagtac atcagcgatc acctggcaga 7260
cagcaatttt gatatcgcca gcgtcgcaca gcatgtttgc ttgtcgccgt cgcgtctgtc 7320
acatcttttc cgccagcagt tagggattag cgtcttaagc tggcgcgagg accaacgtat 7380
cagccaggcg aagctgcttt tgagcaccac ccggatgcct atcgccaccg tcggtcgcaa 7440
tgttggtttt gacgatcaac tctacttctc gcgggtattt aaaaaatgca ccggggccag 7500
cccgagcgag ttccgtgccg gttgtgaaga aaaagtgaat gatgtagccg tcaagttgtc 7560
ataataaatc gatgcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 7620
atttttctaa atacagtcga caacgcagga a 7651
<210> 5
<211> 22
<212> DNA/RNA
<213> pKKma plasmid-upstream primer (Unknown)
<400> 5
gatcctccaa ttcgccctat ag 22
<210> 6
<211> 25
<212> DNA/RNA
<213> pKKma plasmid-downstream primer (Unknown)
<400> 6
ctaatactag cgacgccatc tatgt 25
<210> 7
<211> 29
<212> DNA/RNA
<213> pCasPA plasmid-upstream primer (Unknown)
<400> 7
cggggtacct tcctgcgttg tcgactgta 29
<210> 8
<211> 45
<212> DNA/RNA
<213> pCasPA plasmid-downstream primer (Unknown)
<400> 8
cgaaattcct ttttttccat tttttataac ctccttagag ctcga 45
<210> 9
<211> 25
<212> DNA/RNA
<213> pKKma plasmid-upstream primer (Unknown)
<400> 9
ttgtgaagca acttttgtta ttgtg 25
<210> 10
<211> 29
<212> DNA/RNA
<213> pKKma plasmid-downstream primer (Unknown)
<400> 10
aactgcaggg aggatccggt ctaacaaag 29
<210> 11
<211> 29
<212> DNA/RNA
<213> upstream primer (Unknown)
<400> 11
cggggtacct tcctgcgttg tcgactgta 29
<210> 12
<211> 29
<212> DNA/RNA
<213> downstream primer (Unknown)
<400> 12
aactgcaggg aggatccggt ctaacaaag 29
Claims (1)
1. A novel transposon system for constructing a mutant library, wherein: the construction steps are as follows:
said step (c) is
The transposon plasmid vector has the following characteristics:
The nucleotide sequence of the pKKma transposon plasmid is SEQ ID NO.1;
said step (c) is
IntnpAThe nucleotide sequence of the gene of the transposase is SEQ ID NO.3;
said step (c) is
The nucleotide sequence of the novel transposon plasmid vector is SEQ ID NO.4;
said step (c) is
In which transposon plasmid vector is transformedE .coli Obtaining transposase gene which is stably expressed and regulated by arabinose from S17-1 lambda pirtnpAThe escherichia coli strain of (1) is then used for obtaining a transposon mutant strain of other species containing the transposon by a common conjugal transfer mating method;
said step (c) is
The bacterial concentration and the volume of the transposon mutant strain can be controlled according to the requirement of constructing a library, and the process of transposase inducing transposon jumping can be regulated and controlled;
said step (c) is
Middle coded arabinose promoter genearaBADAnd arabinose inhibitory protein genearaCThe nucleotide sequence of (A) is SEQ ID NO.2;
the method enables a whole genome transposon library of multiple gene insertion patterns covering a whole gene.
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