CN110551749A - Construction method and application of pseudomonas putida suicide vector - Google Patents
Construction method and application of pseudomonas putida suicide vector Download PDFInfo
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Abstract
The invention discloses a construction method of a pseudomonas putida suicide vector and provides an application method of the suicide vector in gene knockout of pseudomonas putida. Firstly, replacing a replicon of a pBBR1MCS-5 plasmid with an R6K replicon to ensure that the replicon can replicate in Escherichia coli S17/lambda pir but not in Pseudomonas putida, and then sequentially inserting an upstream homology arm of a target gene to be knocked out, an antibiotic resistance screening gene and a downstream homology arm of the target gene to be knocked out into the plasmid to obtain the pseudomonas putida gene knockout suicide vector. The suicide vector is introduced into pseudomonas putida through transformation or conjugative transfer, and a deletion mutant strain of a target gene to be knocked out is constructed. The method for constructing the pseudomonas putida gene deletion mutant strain provided by the invention is convenient and rapid, and has high accuracy.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a construction method and application of a pseudomonas putida suicide vector.
Background
Pseudomonas putida is a gram-negative bacterium, has wide metabolic diversity and strong adaptability to different environments, is often used for the research of basic metabolic pathways of bacteria, and is also widely applied to various biotechnological applications, such as the bioremediation of pollutants, the production of special chemical compositions and the like. The gene knockout is a main mode of genetic operation of strains, and is also a main method for researching gene and protein functions and explaining biodegradation ways and regulation, so that the development of an efficient, rapid and accurate gene knockout means is an important prerequisite for fully utilizing pseudomonas putida.
Suicide plasmids refer to plasmids that replicate autonomously in certain bacteria but not in others, and cannot replicate because the replication proteins required for initiation of the replication genes are not present in most bacteria, and thus, when the suicide plasmid enters the host cell, it either cannot replicate, is eliminated, or is integrated into the host chromosome under the action of an external selective pressure, and replicates together with the chromosome. According to the characteristics of suicide plasmid, gene mutation DNA fragments constructed by gene engineering technology are cloned into suicide plasmid, homologous exchange is carried out between homologous fragments at two ends of mutant gene and genome, and an accurate gene deletion mutant strain is constructed, wherein plasmid disappears from thalli along with passage of bacteria due to suicide characteristic and original wild type gene on chromosome.
the currently widely used pseudomonas putida gene knockout vector is pK18mobsacB, and the traditional suicide vector has some obvious defects when gene knockout is carried out, including: (1) sacB is a lethal gene widely used as a suicide vector, but the sucrose lethal effect caused by sacB is weak in Pseudomonas putida, so that the proportion of correctly deleted mutant strains is very low, and the screening workload is increased; (2) the suicide vector is large, so that the conjugation and transfer efficiency of the plasmid is low; (3) the homology arms are connected together for two rounds of screening, but the probability of the second round of exchange reversion to wild type is very high, the accuracy is low, the common means is PCR verification, and the cost for screening the knockout mutant is increased. Thus, existing suicide vectors exhibit significant limitations when used for pseudomonas putida gene knockout.
Disclosure of Invention
In view of the above, the invention provides a construction method and application of a pseudomonas putida suicide vector, which can efficiently, quickly and accurately knockout pseudomonas putida.
The technical scheme of the invention is realized as follows:
in a first aspect, the invention provides a construction method of a pseudomonas putida suicide vector, which comprises the following steps:
S1, reversely amplifying to obtain a DNA fragment by taking the plasmid pBBR1MCS-5 as a template, wherein the nucleotide sequence of the DNA fragment is shown as SEQ ID No. 1;
S2, amplifying by taking pTnmod-RKm' plasmid as a template to obtain an R6K replicon, wherein the nucleotide sequence of the replicon is shown as SEQ ID No. 2;
S3, digesting the DNA fragment obtained in the step S1 and the R6K replicon obtained in the step S2 by using restriction enzymes NdeI, and then connecting to obtain a vector pBR 6K;
S4, amplifying to obtain an upstream homology arm of a target gene x to be knocked out by taking the total DNA of the pseudomonas putida genome as a template, and then connecting to a vector pBR6K to obtain a vector pBR 6K-xup;
s5, amplifying to obtain kanamycin resistance gene by taking pTnmod-RKm' plasmid as a template, and then connecting to a vector pBR6K to obtain a vector pBR 6K-xup-kan;
S6, amplifying to obtain a downstream homology arm of a target gene x to be knocked out by using the total DNA of the pseudomonas putida genome as a template, and then connecting to a vector pBR6K to obtain a vector pBR 6K-xup-kan-down;
In the above technical scheme, the R6K replicon can replicate in Escherichia coli S17/lambda pir and cannot replicate in Pseudomonas putida. An antibiotic resistance screening gene is inserted between the upstream and downstream homology arms of a target gene to be knocked out, on one hand, the antibiotic resistance screening gene is used as a resistance marker site for screening a positive mutant strain, on the other hand, the antibiotic resistance screening gene can be used as a mutation insertion sequence of the target gene to be knocked out, and the antibiotic resistance screening gene interrupts the expression of the target gene.
On the basis of the technical scheme, preferably, the lengths of the upstream homology arm and the downstream homology arm of the target gene x to be knocked out are 600bp-1000bp respectively, so that homologous exchange is easy to occur in a receptor strain, and the efficiency is high.
in a second aspect, the invention provides a method for constructing a pseudomonas putida gene deletion strain by using the pseudomonas putida suicide vector constructed by the method, the pseudomonas putida suicide vector is transformed and introduced into a donor bacterium, then the donor bacterium is mixed with a recipient bacterium, the pseudomonas putida suicide vector is transferred into the recipient bacterium through conjugation, and the strain with the deletion of a target gene to be knocked out is obtained through the co-screening of an antibiotic corresponding to the antibiotic resistance screening gene and gentamicin.
In a third aspect, the invention also provides a method for constructing a pseudomonas putida gene deletion strain by using the pseudomonas putida suicide vector constructed by the method, the pseudomonas putida suicide vector is directly introduced into pseudomonas putida through thermal shock transformation or electric transformation to be cultured to generate homologous exchange, and the strain with the deletion of the target gene to be knocked out is obtained through the co-screening of the antibiotic corresponding to the antibiotic resistance screening gene and gentamicin.
In a fourth aspect, the invention provides an application of the pseudomonas putida suicide vector constructed by the method in gene knock-out of pseudomonas putida.
compared with the prior art, the construction method and the application of the pseudomonas putida suicide vector have the following beneficial effects:
The invention replaces the replicon of the plasmid pBBR1MCS-5 with an R6K replicon, wherein the R6K replicon comes from a phage genome and can replicate in cells under the condition of the existence of a specific protein for identifying the replicon, Escherichia coli S17/lambda pir contains the replication system, but not in Pseudomonas putida, so that a suicide vector is constructed in an S17/lambda pir strain and then is introduced into the Pseudomonas putida, and the suicide vector cannot replicate in the Pseudomonas putida, so that the vector integrated on the genome only can be remained by homologous exchange;
2. The suicide vector disclosed by the invention is small in molecular weight, has mob genes, can be introduced into a receptor strain in a mode of amphiphilic basic conjugation and also in a mode of electric transformation, and is favorable for improving the frequency of conjugation transfer or transformation and the probability of correct integration;
3. The mutant screening can adopt a PCR method and an antibiotic method, the operation is simple, and the accuracy is high;
4. By using the suicide vector of the invention, studies such as gene knock-in and gene mutation can be realized by adopting the same strategy as gene knock-out.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the construction of the vector pBR6K of example 1.
FIG. 2 is a map of the suicide vector pBR6K-fleQup-kan-down of the fleQ gene of example 2.
FIG. 3 is a diagram of the detection of mutants by PCR amplification using the internal primer of fleQ gene in example 2, wherein lane 1 is the amplification product using wild type KT2440 as a template, lanes 2 and 3 are the amplification products using fleQ mutant obtained by screening as a template, and M represents DNA Marker.
FIG. 4 is a diagram of the detection of mutants by PCR amplification using primers outside the fleQ gene in example 2, wherein lane 1 is an amplification product using wild-type KT2440 as a template, lanes 2 and 3 are amplification products using screened fleQ mutants as templates, and M represents DNA Marker.
FIG. 5 is a graph showing the restriction enzyme digestion of the PCR product amplified with the fleQ gene outer primer in example 2 with EcoRI + BamHI, lanes 1, 3 and 5 are amplification products of wild type KT2440 and 2 mutants, respectively, which were not restricted, lanes 2, 4 and 6 are restriction products in lanes 1, 3 and 5, respectively, and M represents DNA Marker.
FIG. 6 is a graph showing the results of examining the motility of the deletion mutant and the complementing strain of fleQ in example 2, wherein WT represents wild-type Pseudomonas putida KT2440, Δ fleQ represents the fleQ mutant, and c Δ fleQ represents the complementing strain of the fleQ mutant.
FIG. 7 is a map of the suicide vector pBR6K-PP _0914up-kan-down of the PP _0914 gene of example 3.
FIG. 8 is a diagram showing the detection of mutants by PCR amplification using the inner primers of PP _0914 gene in example 3, wherein lane 1 shows the amplified product using wild type KT2440 as a template, lanes 2, 3 and 4 show the amplified products using the mutants as a template, and M represents DNA Marker.
FIG. 9 is a diagram showing the detection of mutants by PCR amplification using primers outside the PP _0914 gene in example 3, wherein lane 1 shows the amplification product of wild type KT2440 as a template, lanes 2, 3 and 4 show the amplification products of mutants as templates, and M represents DNA Marker.
FIG. 10 is a graph showing the restriction enzyme digestion of the PCR product amplified with the outer primer of PP _0914 gene with EcoRI + BamHI in example 3, lanes 1, 3, 5 and 7 are the amplification products of wild type KT2440 and 2 mutants which were not restricted by enzyme digestion, lanes 2, 4, 6 and 8 are the restriction enzyme digestion products in lanes 1, 3, 5 and 7, respectively, and M represents DNA Marker.
FIG. 11 is a graph of the detection of the deletion mutant of PP _0914 and the biofilm of wild-type KT2440 in example 3, WT for wild-type Pseudomonas putida KT2440 and. DELTA.PP _0914 for the PP _0914 mutant.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental procedures in the following examples were carried out by a conventional method unless otherwise specified, and the experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The experimental E.coli S17/lambda pir and Pseudomonas putida KT2440 were purchased from Beijing Quanjin and grown in conventional LB medium, selecting the concentrations of antibiotics used for growth as follows: 20. mu.g/mL gentamicin Gm or 50. mu.g/mL kanamycin Km. The original plasmids pBBR1MCS-5 and pTnmod-RKm' used in the experiments were purchased from biotech companies and are commonly available on the market. The detailed steps of vector cloning, conjugal transfer, electrotransformation and competent cell preparation are shown in the molecular cloning experimental guidelines.
example 1 construction of vector pBR6K
The construction scheme of the vector pBR6K is shown in FIG. 1, and the replicon sequence is reversely amplified and removed by using a primer pair P1/P2 and pBBR1MCS-5 plasmid as a template; amplifying the R6K replicon by using a primer pair P3/P4 and pTnmod-RKm' plasmid as a template; both PCR products are cut by restriction enzyme NdeI; then, T4DNA ligase of Takara company is used for connecting the segments after enzyme digestion together; and (5) sequencing verification is carried out, and the R6K replicon is confirmed to be correctly inserted into the vector, so that the vector pBR6K is finally obtained. In the vector pBR6K shown in figure 1, gentamicin-R is a gentamicin resistance gene for screening, and lacZalpha gene codes galactosidase subunit for blue and white class screening; the mob gene allows the plasmid to be transferred in different strains by conjugation of the two parents; there are multiple cloning sites in the lacZ gene, through which the homology arms of the target gene can be ligated into the vector pBR 6K.
Example 2 knock-out of the fleQ Gene of Pseudomonas putida KT2440
S1 construction of suicide vector pBR6K-fleQup-kan-down
FleQ is a transcription regulator in pseudomonas and has important influence on biofilm formation and motility of pseudomonas, and the feasibility of the invention is tested by using a FleQ gene as a target gene. Firstly, taking total DNA of a genome of pseudomonas putida KT2440 as a template, amplifying a homology arm with the upstream of 600bp of a fleQ gene by using a primer pair P5/P6, carrying out enzyme digestion by using XhoI and EcoRI, connecting the enzyme digestion into a pBR6K vector to obtain a vector pBR6K-fleQup, and carrying out sequencing verification; then, using pTnmod-RKm' plasmid as a template, amplifying a kanamycin resistance gene kan by using a primer pair P7/P8, cutting the kanamycin resistance gene kan by using EcoRI and BamHI enzyme, connecting the EcoRI and BamHI enzyme into a pBR6K vector to obtain a vector pBR6K-fleQup-kan, and carrying out sequencing verification; and finally, using total DNA of a pseudomonas putida KT2440 genome as a template, amplifying a homologous arm of 1000bp at the downstream of the fleQ gene by using a primer pair P9/P10, carrying out enzyme digestion by using BamHI and XbaI, connecting the enzyme digestion into a pBR6K vector to obtain a vector pBR6K-fleQup-kan-down, and carrying out sequencing verification to finally obtain a suicide vector pBR 6K-fleQup-kan-down. The map of the suicide vector pBR6K-fleQup-kan-down is shown in FIG. 2.
S2 introduction of suicide vector into Pseudomonas putida KT2440
A suicide vector pBR6K-fleQup-kan-down was introduced into competent cells of the donor bacterium Escherichia coli S17/lambda pir by heat shock transformation or electric transformation, the suicide vector introduced Escherichia coli S17/lambda pir was mixed with the recipient bacterium Pseudomonas putida KT2440, the mixture was shaken at 28 ℃ for 6 hours, and the resulting mixture was spread on an LB plate containing kanamycin (50. mu.g/mL) and allowed to stand for 2 days.
S3, screening and verification of fleQ mutant
Transformants were selected by antibiotic lethal method. If single exchange occurs, the fleQ gene cannot be completely knocked out, and the suicide plasmid can be completely integrated on the genome, so that the suicide plasmid can simultaneously have two resistances of kanamycin and gentamicin; if a double crossover occurs, the fleQ is completely replaced by kan, the suicide plasmid is not integrated into the genome and it cannot replicate in KT2440 and is eliminated in the passage, so the correct fleQ deletion mutant has kanamycin resistance but no gentamycin resistance. Colonies on the plate were picked and inoculated into both of the LB medium containing kanamycin and the LB medium containing kanamycin plus gentamicin, and after 12 hours of culture at 28 ℃, it was observed that the strain which grew only in the medium containing kanamycin but not in the LB containing gentamicin was the correct fleQ mutant.
The mutants were detected by PCR. The resulting mutant is a mutant in which the target gene is replaced with a kanamycin resistance gene. If single crossover occurs, the fleQ gene still exists in the strain, and a band can be amplified by using a fleQ gene inner primer pair P11/P12; if double crossover occurs, the fleQ is completely replaced by kan, and no band can be amplified by using the internal primer pair P11/P12 of the fleQ gene, as shown in FIG. 3, the wild type can amplify a product as a template, and the mutant can amplify no product as a template. In addition, when the fleQ gene outer primer pair P13/P14 was used for detection, single crossover could not amplify the product, and correct double crossover and wild type could amplify the product, as shown in FIG. 4, with wild type and two mutant strains. When the wild type strain and the mutant genome are used as templates, amplified products have different restriction enzyme sites, EcoRI and BamHI restriction enzyme sites do not exist in the wild type, and the presence of the mutant can be detected by using an enzyme cutting mode to determine whether the mutant is correct. As shown in FIG. 5, the wild-type PCR product could not be cleaved and the mutant could be cleaved.
S4 and detecting mobility of fleQ mutant
The fleQ gene plays an important role in regulating and controlling the synthesis process of pseudomonas putida flagella, the strain loses mobility due to the fact that the fleQ gene is knocked out, and the mobility detection is carried out on the fleQ mutant screened by the method. As shown in FIG. 6, consistent with the results reported, the fleQ mutant has no motility after 12 hours of static culture, and the motility is partially recovered after complementation, which indicates that the strain obtained by the method of the present invention is a correct fleQ knockout mutant.
Example 3 knock-out of the PP _0914 Gene of Pseudomonas putida KT2440
S1 construction of suicide vector pBR6K-PP _0914up-kan-down
PP _0914 is a phosphodiesterase in Pseudomonas putida KT2440 and is responsible for degrading the second messenger molecule c-di-GMP, regulating the biofilm and motility of the strain, and the deletion of PP _0914 leads to the enhancement of the biofilm forming capacity of the strain. The present invention uses the PP _0914 gene as a target gene to test the feasibility of the present invention. Firstly, taking total DNA of a genome of pseudomonas putida KT2440 as a template, amplifying a homology arm of 1000bp upstream of a pp _0914 gene by using a primer pair P15/P16, then connecting the homology arm into a pBR6K vector by using XhoI + EcoRI, and carrying out sequencing verification; then, using pTnmod-RKm' plasmid as a template, amplifying a kanamycin resistance gene kan by using a primer pair P7/P8, cutting the kanamycin resistance gene kan by using EcoRI and BamHI enzyme, and connecting the EcoRI and BamHI enzyme into a pBR6K vector for sequencing verification; and finally, using total DNA of a pseudomonas putida KT2440 genome as a template, amplifying a homologous arm of 600bp at the downstream of the PP _0914 gene by using a primer pair P17/P18, carrying out enzyme digestion by using BamHI and XbaI, connecting the enzyme digestion into a pBR6K vector, and carrying out sequencing verification to finally obtain a suicide vector pBR6K-PP _0914 up-kan-down. The map of the suicide vector pBR6K-PP _0914up-kan-down is shown in FIG. 7.
s2 introduction of suicide vector into Pseudomonas putida KT2440
The suicide vector pBR6K-PP _0914up-kan-down was introduced into Pseudomonas putida KT2440 competent cells by heat shock transformation or electric transformation, and after thawing in a shaker at 28 ℃ for 6 hours, the bacterial solution was spread on LB plate containing kanamycin (50. mu.g/mL) and cultured for 2 days with standing.
Screening and validation of S3 and PP _0914 mutants
transformants were selected by antibiotic lethal method. The correct PP _0914 mutant was screened using the same antibiotic lethal protocol as step S3 of example 2.
the mutants were detected by PCR. If single crossover occurs, the PP _0914 gene still exists in the strain, and a strip can be amplified by using the PP _0914 gene inner primer pair P19/P20; if double crossover occurs, PP _0914 is completely replaced by kan, and no band can be amplified by using the PP _0914 gene inner primer pair P19/P20, as shown in FIG. 8, the wild type can be used as a template to amplify a product, and the mutant is used as a template to amplify no product. In addition, when the PP _0914 gene outer primer pair P21/P22 was used for detection, single crossover failed to amplify the product, and correct double crossover and wild type amplified the product, as shown in FIG. 9, with wild type and two mutant strains. When the wild type strain and the mutant genome are used as templates, amplified products have different restriction enzyme sites, EcoRI and BamHI restriction enzyme sites do not exist in the wild type, and the presence of the mutant can be detected by using an enzyme cutting mode to determine whether the mutant is correct. As shown in FIG. 10, the wild-type PCR product could not be cleaved and the mutant could be cleaved.
S4, PP _0914 biological quilt shaping ability test
When the wild strain and the mutant strain are respectively cultured in a glass tube for 12 hours, the biofilm formed by the PP _0914 mutant is obviously more than that formed by wild KT2440, and as shown in figure 11, the PP _0914 deletion mutant constructed by the invention is correct.
TABLE 1 primer sequences used in the present invention
| Primer and method for producing the same | DNA Sequence (5 '-3') | Sequence numbering |
| P1 | ggaattccatatgcctcgctaacggattcacc | SEQ ID No.3 |
| P2 | ggaattccatatgtttcgcatttcgccctat | SEQ ID No.4 |
| P3 | ggaattccatatgcatgcatgtcgacggtac | SEQ ID No.5 |
| P4 | ggaattccatatgcacaacgtggctttccc | SEQ ID No.6 |
| P5 | ccggaattctcagcaccaccgcaagg | SEQ ID No.7 |
| P6 | ccgctcgagtgaagccactccgcacc | SEQ ID No.8 |
| P7 | cgcggatcccctcgtgaagaaggtgttg | SEQ ID No.9 |
| P8 | ccggaattcaagccacgttgtgtctcaa | SEQ ID No.10 |
| P9 | tgctctagacttcaaggtcgcggcga | SEQ ID No.11 |
| P10 | cgcggatcccgtattcgccgtaccacc | SEQ ID No.12 |
| P11 | attgaagcgaatggaaccg | SEQ ID No.13 |
| P12 | gctgctggaaagcgaactg | SEQ ID No.14 |
| P13 | ttgcacagcagctcaccc | SEQ ID No.15 |
| P14 | cccttcgccagtaccagaa | SEQ ID No.16 |
| P15 | ccggaattcgcatggcatcattttgcc | SEQ ID No.17 |
| P16 | ccgctcgagccctgcggaacctctatt | SEQ ID No.18 |
| P17 | ctagtctagattgtcgacgtagtcgatc | SEQ ID No.19 |
| P18 | cgcggatcccgggtttcggtgtcctga | SEQ ID No.20 |
| P19 | caccatgcccagttccg | SEQ ID No.21 |
| P20 | tcgaccacgagcaaatcc | SEQ ID No.22 |
| P21 | ctgaagcagtcttgctcgtct | SEQ ID No.23 |
| P22 | cctatgtttgttgcgtggttat | SEQ ID No.24 |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan Boohot Biotechnology Ltd
<120> construction method and application of pseudomonas putida suicide vector
<130> 2019-9-23
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3765
<212> DNA
<213> (Artificial sequence)
<400> 1
catatgcctc gctaacggat tcaccgtttt tatcaggctc tgggaggcag aataaatgat 60
catatcgtca attattacct ccacggggag agcctgagca aactggcctc aggcatttga 120
gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata 180
agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt 240
ctgccattca tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc 300
aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtcg gcctattggt 360
taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 420
caatttccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 480
ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac 540
gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga 600
ctcactatag ggcgaattgg agctccaccg cggtggcggc cgctctagaa ctagtggatc 660
ccccgggctg caggaattcg atatcaagct tatcgatacc gtcgacctcg agggggggcc 720
cggtacccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt 780
catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 840
gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 900
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 960
gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcat gcataaaaac tgttgtaatt 1020
cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca 1080
gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggacgca caccgtggaa 1140
acggatgaag gcacgaaccc agttgacata agcctgttcg gttcgtaaac tgtaatgcaa 1200
gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg 1260
cgcagtggcg gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc 1320
atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga 1380
tgttacgcag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg 1440
gctcaagtat gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc 1500
gggctgctct tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc 1560
agccggactc cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg 1620
ccttcgacca agaagcggtt gttggcgctc tcgcggctta cgttctgccc aggtttgagc 1680
agccgcgtag tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg 1740
gcattgccac cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg 1800
tgatctacgt gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg 1860
gcatacggga agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg 1920
ttcaagccga gatcggcttc ccggccgcgg agttgttcgg taaattgtca caacgccgcc 1980
aggtggcact tttcggggaa atgtgcgcgc ccgcgttcct gctggcgctg ggcctgtttc 2040
tggcgctgga cttcccgctg ttccgtcagc agcttttcgc ccacggcctt gatgatcgcg 2100
gcggccttgg cctgcatatc ccgattcaac ggccccaggg cgtccagaac gggcttcagg 2160
cgctcccgaa ggtctcgggc cgtctcttgg gcttgatcgg ccttcttgcg catctcacgc 2220
gctcctgcgg cggcctgtag ggcaggctca tacccctgcc gaaccgcttt tgtcagccgg 2280
tcggccacgg cttccggcgt ctcaacgcgc tttgagattc ccagcttttc ggccaatccc 2340
tgcggtgcat aggcgcgtgg ctcgaccgct tgcgggctga tggtgacgtg gcccactggt 2400
ggccgctcca gggcctcgta gaacgcctga atgcgcgtgt gacgtgcctt gctgccctcg 2460
atgccccgtt gcagccctag atcggccaca gcggccgcaa acgtggtctg gtcgcgggtc 2520
atctgcgctt tgttgccgat gaactccttg gccgacagcc tgccgtcctg cgtcagcggc 2580
accacgaacg cggtcatgtg cgggctggtt tcgtcacggt ggatgctggc cgtcacgatg 2640
cgatccgccc cgtacttgtc cgccagccac ttgtgcgcct tctcgaagaa cgccgcctgc 2700
tgttcttggc tggccgactt ccaccattcc gggctggccg tcatgacgta ctcgaccgcc 2760
aacacagcgt ccttgcgccg cttctctggc agcaactcgc gcagtcggcc catcgcttca 2820
tcggtgctgc tggccgccca gtgctcgttc tctggcgtcc tgctggcgtc agcgttgggc 2880
gtctcgcgct cgcggtaggc gtgcttgaga ctggccgcca cgttgcccat tttcgccagc 2940
ttcttgcatc gcatgatcgc gtatgccgcc atgcctgccc ctcccttttg gtgtccaacc 3000
ggctcgacgg gggcagcgca aggcggtgcc tccggcgggc cactcaatgc ttgagtatac 3060
tcactagact ttgcttcgca aagtcgtgac cgcctacggc ggctgcggcg ccctacgggc 3120
ttgctctccg ggcttcgccc tgcgcggtcg ctgcgctccc ttgccagccc gtggatatgt 3180
ggacgatggc cgcgagcggc caccggctgg ctcgcttcgc tcggcccgtg gacaaccctg 3240
ctggacaagc tgatggacag gctgcgcctg cccacgagct tgaccacagg gattgcccac 3300
cggctaccca gccttcgacc acatacccac cggctccaac tgcgcggcct gcggccttgc 3360
cccatcaatt tttttaattt tctctgggga aaagcctccg gcctgcggcc tgcgcgcttc 3420
gcttgccggt tggacaccaa gtggaaggcg ggtcaaggct cgcgcagcga ccgcgcagcg 3480
gcttggcctt gacgcgcctg gaacgaccca agcctatgcg agtgggggca gtcgaaggcg 3540
aagcccgccc gcctgccccc cgagcctcac ggcggcgagt gcgggggttc caagggggca 3600
gcgccacctt gggcaaggcc gaaggccgcg cagtcgatca acaagccccg gaggggccac 3660
tttttgccgg agggggagcc gcgccgaagg cgtgggggaa ccccgcaggg gtgcccttct 3720
ttgggcacca aagaactaga tatagggcga aatgcgaaac atatg 3765
<210> 2
<211> 499
<212> DNA
<213> (Artificial sequence)
<400> 2
catatgcatg catgtcgacg gtaccccccc atgtcagccg ttaagtgttc ctgtgtcact 60
caaaattgct ttgagaggct ctaagggctt ctcagtgcgt tacatccctg gcttgttgtc 120
cacaaccgtt aaaccttaaa agctttaaaa gccttatata ttcttttttt tcttataaaa 180
cttaaaacct tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg 240
agagcttagt acgtgaaaca tgagagctta gtacgttagc catgagagct tagtacgtta 300
gccatgaggg tttagttcgt taaacatgag agcttagtac gttaaacatg agagcttagt 360
acgtgaaaca tgagagctta gtacgtacta tcaacaggtt gaactgctga tcttcagatc 420
ctctacgccg gacgcatcgt ggccggggtt cgaaatcgat gagctcgggg ggggggggga 480
aagccacgtt gtgcatatg 499
<210> 3
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 3
ggaattccat atgcctcgct aacggattca cc 32
<210> 4
<211> 31
<212> DNA
<213> (Artificial sequence)
<400> 4
ggaattccat atgtttcgca tttcgcccta t 31
<210> 5
<211> 31
<212> DNA
<213> (Artificial sequence)
<400> 5
ggaattccat atgcatgcat gtcgacggta c 31
<210> 6
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 6
ggaattccat atgcacaacg tggctttccc 30
<210> 7
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 7
ccggaattct cagcaccacc gcaagg 26
<210> 8
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 8
ccgctcgagt gaagccactc cgcacc 26
<210> 9
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 9
cgcggatccc ctcgtgaaga aggtgttg 28
<210> 10
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 10
ccggaattca agccacgttg tgtctcaa 28
<210> 11
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 11
tgctctagac ttcaaggtcg cggcga 26
<210> 12
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 12
cgcggatccc gtattcgccg taccacc 27
<210> 13
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 13
attgaagcga atggaaccg 19
<210> 14
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 14
gctgctggaa agcgaactg 19
<210> 15
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 15
ttgcacagca gctcaccc 18
<210> 16
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 16
cccttcgcca gtaccagaa 19
<210> 17
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 17
ccggaattcg catggcatca ttttgcc 27
<210> 18
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 18
ccgctcgagc cctgcggaac ctctatt 27
<210> 19
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 19
ctagtctaga ttgtcgacgt agtcgatc 28
<210> 20
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 20
cgcggatccc gggtttcggt gtcctga 27
<210> 21
<211> 17
<212> DNA
<213> (Artificial sequence)
<400> 21
caccatgccc agttccg 17
<210> 22
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 22
tcgaccacga gcaaatcc 18
<210> 23
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 23
ctgaagcagt cttgctcgtc t 21
<210> 24
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 24
cctatgtttg ttgcgtggtt at 22
Claims (5)
1. A construction method of a pseudomonas putida suicide vector is characterized by comprising the following steps:
s1, reversely amplifying to obtain a DNA fragment by taking the plasmid pBBR1MCS-5 as a template, wherein the nucleotide sequence of the DNA fragment is shown as SEQ ID No. 1;
S2, amplifying by taking pTnmod-RKm' plasmid as a template to obtain an R6K replicon, wherein the nucleotide sequence of the replicon is shown as SEQ ID No. 2;
S3, digesting the DNA fragment obtained in the step S1 and the R6K replicon obtained in the step S2 by using restriction enzymes NdeI, and then connecting to obtain a vector pBR 6K;
S4, amplifying to obtain an upstream homology arm of a target gene x to be knocked out by taking the total DNA of the pseudomonas putida genome as a template, and then connecting to a vector pBR6K to obtain a vector pBR 6K-xup;
s5, amplifying to obtain kanamycin resistance gene by taking pTnmod-RKm' plasmid as a template, and then connecting to a vector pBR6K to obtain a vector pBR 6K-xup-kan;
s6, using the total DNA of the pseudomonas putida genome as a template, amplifying to obtain a downstream homologous arm of the target gene x to be knocked out, and then connecting to the vector pBR6K to obtain the vector pBR 6K-xup-kan-down.
2. The method of constructing a pseudomonas putida suicide vector according to claim 1, wherein: the lengths of the upstream homology arm and the downstream homology arm of the target gene x to be knocked out are respectively 600bp-1000 bp.
3. A method for constructing a Pseudomonas putida gene deletion strain using the Pseudomonas putida suicide vector constructed by the method of claim 1, wherein: and (2) transferring the pseudomonas putida suicide vector into donor bacteria through transformation, then mixing the donor bacteria with acceptor bacteria, transferring the pseudomonas putida suicide vector into the acceptor bacteria through conjugal transfer, and obtaining the strain with the deletion of the target gene to be knocked out through common screening of kanamycin and gentamicin.
4. a method for constructing a Pseudomonas putida gene deletion strain using the Pseudomonas putida suicide vector constructed by the method of claim 1, wherein: and directly converting the pseudomonas putida suicide vector into pseudomonas putida for culture to generate homologous exchange, and jointly screening by kanamycin and gentamicin to obtain the strain with the deletion of the target gene to be knocked out.
5. Use of a pseudomonas putida suicide vector constructed by the method of claim 1 in gene knock-out of pseudomonas putida.
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| CN112280724A (en) * | 2020-10-27 | 2021-01-29 | 西安交通大学 | Pseudomonas putida ND6 coupled protein complex dotN gene deletion mutant containing resistance marker, and construction method and determination method thereof |
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| CN112280724A (en) * | 2020-10-27 | 2021-01-29 | 西安交通大学 | Pseudomonas putida ND6 coupled protein complex dotN gene deletion mutant containing resistance marker, and construction method and determination method thereof |
| CN112342177A (en) * | 2020-10-27 | 2021-02-09 | 西安交通大学 | Pseudomonas putida ND6 coupled protein complex dotM gene deletion mutant strain and construction method and determination method thereof |
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| CN116286934A (en) * | 2022-12-23 | 2023-06-23 | 江南大学 | Research method for regulating tolerance of organic compounds by cold shock protein in pseudomonas putida |
| CN116286934B (en) * | 2022-12-23 | 2024-07-12 | 江南大学 | Research method for regulating tolerance of organic compounds by cold shock protein in pseudomonas putida |
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Application publication date: 20191210 |