阅读说明：本技术 一种恶臭假单胞菌***载体的构建方法及其应用 (Construction method and application of pseudomonas putida suicide vector ) 是由 崔格特 于 2019-10-09 设计创作，主要内容包括：本发明公开了一种恶臭假单胞菌自杀载体的构建方法,并提供了该自杀载体在恶臭假单胞菌中进行基因敲除的应用方法。首先将pBBR1MCS-5质粒的复制子替换成R6K复制子,使得其能在大肠杆菌S17/λpir中复制,而不能在恶臭假单胞菌中复制,然后在该质粒中依次插入待敲除靶基因的上游同源臂、抗生素抗性筛选基因和待敲除靶基因的下游同源臂,从而得到恶臭假单胞菌基因敲除自杀载体。将该自杀载体通过转化或接合转移导入恶臭假单胞菌中,构建待敲除靶基因的缺失突变株。本发明提出的构建恶臭假单胞菌基因缺失突变株的方法,方便快捷,正确率高。(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.)

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.

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.