CN120118931A - A plasmid system for scarless gene editing and drug-resistant plasmid elimination in Enterobacteriaceae and its application - Google Patents

Abstract

The present invention belongs to the field of genetic engineering, and specifically relates to a plasmid system for traceless gene editing and drug-resistant plasmid removal in Enterobacteriaceae and its application. The present invention provides a set of multifunctional tool plasmid systems that can simultaneously quickly edit genes in Enterobacteriaceae and remove plasmids, including a pGGTOX plasmid with gene editing and plasmid removal, and a pCP‑oriT plasmid that eliminates resistance genes to achieve traceless gene knockout. The principle of homologous recombination can be used to edit genes and eliminate plasmids in Enterobacteriaceae within 3‑4 days at the fastest, providing a new idea for the construction of a multifunctional tool plasmid system, and has good application prospects.

Description

Plasmid system for traceless gene editing and drug-resistant plasmid removal in enterobacteriaceae bacteria and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a plasmid system for traceless gene editing and drug-resistant plasmid removal in enterobacteriaceae bacteria.

Background

Homologous recombination is an important genetic phenomenon in organisms and depends on the homology of DNA sequences. In cells, when DNA fragments of similar sequence are present, they can be exchanged and recombined by the action of a series of enzymes. The naturally occurring process provides a theoretical basis for gene editing by homologous recombination. By designing a DNA fragment containing sequences homologous to both sides of the target gene and introducing it into cells, it is possible to direct the mechanism of homologous recombination within the cells to precisely modify the target gene, such as knockout, substitution or insertion of a specific gene sequence.

With the continued penetration of life science research, precise gene editing techniques are becoming increasingly important. In the fields of medicine, agriculture, biotechnology, etc., it is required to modify specific genes to study functions thereof, treat diseases, improve crop varieties, etc. The high-precision operation of the gene can be realized by utilizing homologous recombination to edit the gene, and the uncertainty brought by some random insertion or mutation methods is avoided.

The spread of bacterial resistance presents a great threat to public health. The transfer of drug-resistant plasmids between bacteria is one of the main routes of drug resistance diffusion. The generation and transmission of drug-resistant bacteria can be effectively reduced by knocking out drug-resistant plasmids. By utilizing the principle of homologous recombination, homologous recombination fragments aiming at specific areas on the drug-resistant plasmid can be designed, and a recombination mechanism in cells is guided to remove key genes or sequences on the drug-resistant plasmid, so that the knockout of the drug-resistant plasmid is realized.

Continuous advances in molecular biology techniques, such as gene cloning, vector construction, nuclease technology, etc., have provided technical support for constructing tool plasmids based on homologous recombination. By using these techniques, researchers can integrate elements required for homologous recombination, such as a homology arm, a selection marker, etc., into a plasmid vector to construct a tool plasmid capable of efficiently performing gene editing and drug-resistant plasmid knockout in cells. Meanwhile, various advanced genetic manipulation techniques, such as electroporation, transformation, etc., have made it easier and more efficient to introduce tool plasmids into cells.

At present, the CRISPR/Cas9 system is widely applied in the field of gene editing, but the CRISPR/Cas9 technology has the problem of nonspecific shearing, namely, shearing is possibly performed at a place beyond a target site, so that off-target effect is caused. This can affect the efficiency and accuracy of editing. And the CRISPR/Cas9 system has complicated tool construction and long time consumption in the application process, so that a multifunctional gene editing tool with high efficiency and high accuracy is urgently needed in the field of gene editing.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a plasmid system for traceless gene editing and drug-resistant plasmid removal in enterobacteriaceae bacteria and application thereof.

In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a plasmid system for traceless gene editing and drug resistant plasmid removal in bacteria of the enterobacteriaceae family, comprising a pGGTOX plasmid having gene editing and plasmid removal and a pCP-oriT plasmid having resistance gene removal, wherein said pGGTOX comprises, in order, an MqsR gene, an R6K replicon, a rhaS gene, a SFGFP gene and an Apr gene, and said pCP-oriT comprises, in order, an FLIP gene, a lambda-repressor gene, a CmR gene, a Rep101 gene, a Hyg gene and an oriT gene.

Further, the Enterobacteriaceae bacteria include one or more of the genus Escherichia, salmonella, shigella, klebsiella, serratia, yersinia, and Proteus, preferably, the method comprises the steps of, the Enterobacteriaceae bacteria include the genus Escherichia and the genus Klebsiella.

In a second aspect, the present invention provides a method for constructing the plasmid system described above, comprising the steps of:

Constructing pGGTOX plasmids, namely respectively amplifying an R6K gene, an Apr gene, an MqsR gene, a rhaS gene and a SFGFP gene to obtain target gene fragments, using PGGASELSCT plasmids as templates, amplifying the amplified large gene fragments by using primers pGGE1 and pGGE2, carrying out phosphorylation and cyclization by using T4 enzyme to obtain pGGA plasmids, inserting the Apr gene into the pGGA plasmids to obtain pGGB plasmids, amplifying the Apr gene with enzyme cutting sites on two sides by using the primers, and connecting the R6K gene, the Apr gene with enzyme cutting sites on two sides, the MqsR gene, the rhaS gene and the SFGFP gene fragments by a one-step method to obtain pGGTOX plasmids;

Constructing pCP-oriT plasmid, namely respectively amplifying the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene to obtain target gene fragment, and connecting the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene into a loop by using seamless cloning technology to obtain pCP-oriT plasmid.

Further, in the pGGTOX plasmid construction step, two pairs of BsmBI and BsaI cleavage sites are introduced flanking the Apr gene.

In a third aspect of the present invention, there is provided a method for gene knockout in bacteria of the enterobacteriaceae family, using the plasmid system of claim for gene knockout, comprising the steps of:

Determining gene fragments to be knocked out in enterobacteriaceae bacteria, amplifying two sides of a gene to be knocked out by designing a primer to obtain two homologous arm gene fragments, inserting the two homologous arm gene fragments into pGGTOX plasmids by a one-step method, converting the two homologous arm gene fragments into carrier bacteria WM3064 to serve as donor bacteria, taking enterobacteriaceae bacteria as acceptor bacteria, performing joint transfer on the donor bacteria and the acceptor bacteria, knocking out the gene fragments to be knocked out by utilizing a homologous recombination principle to obtain a recombinant strain, taking WM3064 transformed with pCP-oriT plasmids as donor bacteria, performing joint transfer on the recombinant strain serving as acceptor bacteria, eliminating the resistant gene fragments with the middle of the homologous arm genes, and removing the pCP-oriT plasmids by increasing the culture temperature.

In a fourth aspect of the present invention, there is provided a method for removing a drug-resistant plasmid from enterobacteriaceae bacteria, wherein the plasmid system removes the drug-resistant plasmid from enterobacteriaceae bacteria, comprising the steps of:

Determining a plasmid to be knocked out in enterobacteriaceae bacteria, amplifying a gene sequence in the plasmid to be knocked out by designing a primer to obtain two homologous arm gene fragments, inserting the two homologous arm gene fragments into pGGTOX plasmids by a one-step method, converting the two homologous arm gene fragments into carrier bacteria WM3064 to serve as donor bacteria, taking enterobacteriaceae bacteria as acceptor bacteria, performing conjugation and transfer on the donor bacteria and the acceptor bacteria, fusing pGGTOX plasmids with the plasmid to be knocked out by utilizing a homologous recombination principle to obtain a recombinant strain, and utilizing rhamnose to induce MqsR toxin gene expression to remove fusion plasmids, so that drug-resistant plasmids are removed.

In a fifth aspect of the present invention there is provided the use of the above plasmid system for gene editing and drug resistant plasmid clearance in bacteria of the enterobacteriaceae family comprising one or more of the genera escherichia, salmonella, shigella, klebsiella, serratia, yersinia, proteus, preferably the enterobacteriaceae family comprising escherichia and klebsiella.

The technical scheme of the invention has the beneficial effects that:

The invention uses the source recombination technology to remove the drug-resistant genes and drug-resistant plasmids in bacteria carrying plasmids. Firstly screening homologous arm sequences on two sides of a targeted drug-resistant gene, ensuring that the homologous recombination of plasmids of pGGTOX and pCP-oriT tools has high specificity and does not affect a host gene, secondly, constructing pGGTOX plasmids with green fluorescent protein, and observing under a fluorescent microscope to show green, thereby rapidly identifying whether pGGTOX plasmids enter a target strain, saving time for screening the target strain, and greatly improving the gene editing efficiency by only 3 days at the shortest time from a wild strain to the knockout of the target gene and the drug-resistant plasmid in the whole homologous recombination process and providing a new tool for removing the drug resistance of antibiotics.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a schematic diagram of a plasmid constructed to be pGGTOX successful in the examples of the present invention;

FIG. 2 is a schematic diagram of a plasmid of pCP-oriT constructed successfully in the examples of the present invention;

FIG. 3 is a diagram illustrating pGGTOX operations in accordance with one embodiment of the present invention;

FIG. 4 is a schematic diagram of an embodiment pGGTOX of an example of the present invention;

FIG. 5 is an electrophoresis diagram of four fragment interfaces after successful pellet construction in a mass embodiment of the present invention;

FIG. 6 shows a successful DAP phenotype knockout chart and a homology arm two-sided electrophoresis chart in an embodiment of the present invention, wherein A is the successful DAP phenotype knockout chart and B is the homology arm two-sided electrophoresis chart;

FIG. 7 is an electrophoretogram of successful knockout mrk of the plasmid according to an example of the present invention;

FIG. 8 is an electrophoresis diagram of a successful knockout of TA gene sequence in an embodiment of the invention;

FIG. 9 is a phenotype diagram of the Apr resistance gene knocked out in the examples of the present invention.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention.

In one embodiment of the present invention, a plasmid system for traceless gene editing and drug resistant plasmid clearance in enterobacteriaceae bacteria is provided, comprising a pGGTOX plasmid with gene editing and plasmid clearance and a pCP-oriT plasmid with resistance gene elimination.

The pGGTOX contains an MqsR gene, an R6K replicon, a rhaS gene, a SFGFP gene and an Apr gene which are connected in sequence.

Specifically, pGGTOX plasmid uses MqsR toxin gene as reverse selection marker, and the gene is not existed in most enterobacteria, and can be used as reverse selection marker of enterobacteria under the control of rhamnose promoter.

The replicon of the pGGTOX plasmid is R6K, which can be replicated in WM3064 recipient bacteria by means of the replication protein provided by pir gene. However, replication is not possible in E.coli, and replication-related proteins must be provided in dependence on pir genes, whereas recipient bacteria do not contain pir genes. Thus, the homologous recombinant strain can be successfully selected.

The pGGTOX plasmid contains SFGFP gene, and the vector strain carrying pGGTOX plasmid can observe green fluorescence under a fluorescence microscope, so that positive clones can be conveniently identified.

Two pairs of BsmBI and BsaI enzyme cutting sites are also introduced into pGGTOX plasmids, and different sticky ends are generated after enzyme cutting, so that the tool plasmids containing two homology arms can be constructed by a one-step method;

the introduction of the apramycin resistance in pGGTOX plasmids is convenient for the gene editing of clinical isolated strains, and is more favorable for the screening of strains with successful homologous recombination.

According to the invention, mqsR genes, R6K replicon, rhaS genes, SFGFP genes and Apr genes are combined for the first time to construct pGGTOX plasmids, and experiments prove that the plasmids can only grow in WM3064 donor bacteria, can be introduced into recipient bacteria to realize the function of gene editing, but cannot exist stably in the recipient bacteria, so that the aim of traceless gene editing can be achieved.

The pCP-oriT contains FLIP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene which are connected in sequence.

Specifically, pCP-oriT has temperature sensitive replicon, and can replicate normally at 30 ℃ and is lost automatically at a temperature higher than 37 ℃, so that plasmid self-elimination after work is facilitated.

The pCP-oriT plasmid contains DNA recombinase gene-FLP recombinase of yeast, can eliminate scars left by gene knockout, and complements gene elimination of pGGTOX plasmid to form a set of traceless gene knockout system.

FLP recombinase of pCP-oriT plasmid recognizes FRT site, deletes resistance gene of FRT-flanked, and realizes traceless knockout.

The pCP-oriT plasmid carries hygromycin and chloramphenicol resistance genes, and the two resistance genes are more favorable for screening the binders after conjugation and transfer with the target strain.

Meanwhile, pGGTOX plasmid can only grow in WM3064 donor bacteria, and is suicide plasmid in acceptor bacteria. The pCP-oriT plasmid replicates normally in the host strain, consistent with the growth curve of the wild type.

Further, the Enterobacteriaceae bacteria include one or more of the genus Escherichia, salmonella, shigella, klebsiella, serratia, yersinia, and Proteus, preferably, the method comprises the steps of, the Enterobacteriaceae bacteria include the genus Escherichia and the genus Klebsiella.

In still another embodiment of the present invention, there is provided a method for constructing the plasmid system described above, comprising the steps of:

Constructing pGGTOX plasmids, namely respectively amplifying an R6K gene, an Apr gene, an MqsR gene, a rhaS gene and a SFGFP gene to obtain target gene fragments, using PGGASELSCT plasmids as templates, amplifying the amplified gene large fragments by using a primer, carrying out phosphorylation and cyclization by using T4 enzyme to obtain pGGA plasmids, inserting the Apr gene into the pGGA plasmids to obtain pGGB plasmids, amplifying the Apr genes with enzyme cutting sites at two sides by using the primer, and connecting the R6K gene, the Apr genes with enzyme cutting sites at two sides, the MqsR gene, rhaS genes and SFGFP gene fragments by using a one-step method to obtain pGGTOX plasmids;

Specifically, the method comprises the following steps:

1. pGGTOX tool preparation of plasmid Pre-construction

First we modified PGGASELECT (the present tool plasmid was purchased from Biolabs, NEW ENGLAND, uk) plasmid. There are multiple type II sites on this plasmid, including (BsmB I, bsa I, etc.). Next, the pSZU-GFP plasmid was used as a template, and the gene sequences for resistance to R6K and Apr contained in the plasmid were amplified by PCR. pSZU941-GFP plasmid is constructed by total gene synthesis in the early stage of the laboratory, and the nucleotide sequence is shown as SEQ ID NO. 1. Finally, we amplified the gene sequences of MqsR, rhaS and SFGFP in the plasmid using pTOX-GFP plasmid with green fluorescence as template. The pTOX-GFP plasmid used in our laboratory was composed of pSZU941-GFP and pTOX (construction process reference: A New Suite of Allelic-Exchange Vectors for THE SCARLESS Modification of Proteobacterial Genomes; article website https:// doi. Org/10.1128/AEM.00990-19) plasmids as templates, and the fragments of interest amplified by primers pTox-GFP-F/pTox-GFP-R and pTox-GFP-F/pTox-GFP-R, respectively, were ligated by ligation, and the ligation interface was identified by primer tsPurpleF/GFP-AC-F, with the identification indicating successful construction of the p' TOX8-GFP tool plasmid.

The primer sequences are as follows:

pTox-GFP-F:GGCCTTCTTCTGATATCGAGCTCTTGACGG;

pTox-GFP-R:TGTACACCTGGATCCCCAGCCTACACAA;

pTox-GFP-F:GGCCTTCTTCTGATATCGAGCTCTTGACGG;

pTox-GFP-R:TGTACACCTGGATCCCCAGCCTACACAA;

tsPurpleF:CGCGACGGTTTCTTACAGTG;

GFP-AC-F:CAGAGTCGGCCAAGGGACCGGCAGTTTACC。

2. PGGASELECT linearization and point mutation of the plasmid realize convenient and directional insertion of a homology arm.

We linearized the PGGASELECT plasmid with PGGASELECT synthetic primers followed by point mutation, then phosphorylates its 5' sticky end with T4 phosphorylase, and then ligates into a loop with T4 ligase, since the two required enzymes Bsa I and BsmB I belong to Type IIs endonucleases, they are able to recognize non-palindromic sequences and cleave outside the recognition sequences. Therefore, the purpose of the modification is to introduce a new gene sequence in the vicinity of the cleavage site of the two enzymes so as to distinguish it from the gene sequence of the original cleavage site, thereby ensuring the specificity in the later gene ligation. Specifically, plasmid PGGASELSCT is used as a template, primers pGGE1 and pGGE2 (recognition sites of restriction enzymes are designed on the primers) are artificially synthesized for amplification, and the amplified large gene fragment is subjected to phosphorylation and then cyclization by using T4 enzyme to obtain a novel plasmid pGGA. The primer sequences are shown below:

pGGE1:CCATCGTCTCACCACGGTCTCACCACTCCTGTAG;

pGGE2:ACTCCCGTCTCGATCCGGTCTCGATCCGTACCAAG。

3. construction of plasmid pGGB for connecting 3-segment Gene fragment by seamless cloning

After point mutation, we artificially synthesized primers pGG-Apr-F, pGG-Apr-R, bsa-BsmB-R, bsa-BsmB-F, amplified the Apr resistance gene in pSZU941-GFP and the backbone and four cleavage sites on pGGA plasmid by PCR, then linearized and used as backbone by using NotI enzyme as a template using pGGA point mutant plasmid as a template, ligated the three fragments by using infusion and sandwiched the Apr resistance gene in the middle of the cleavage sites so that two BsmB I and Bsa I cleavage sites are located on both sides of the Apr resistance gene, and the three fragments form a new plasmid pGGB. The primer sequences are shown below:

pGG-Apr-F:ATTCTCGAGGCGGCCTGTTTGTCGGTGAACGCTCT;

pGG-Apr-R:CTCCGTACCAAGACTTGGGGATCCACTAGTGAGCT;

Bsa-BsmB-R:GACTCACATGCGGCCAGAAGTCTACAGGAATGGTGAGAC;

Bsa-BsmB-F:AGTCTTGGTACGGAGCGAGA。

Construction of pGGTOX tool plasmid

Finally, the R6K gene of pSZU941-GFP plasmid, the gg-Apr-ggF and gg-Apr-ggR of the artificially synthesized primers R6K-oriT-F and R6K-oriT-R are amplified by PCR, the multiple II-type enzyme cutting sites and APr resistance genes of pGGB tool plasmids are amplified by PCR, the rhaS and MqsR genes of pTOX-GFP plasmids and SFGFP genes of the artificially synthesized primers mqsR-F/mqsR-R and rhaS-GFP-F/rhaS-R are amplified by PCR, the four gene fragments are connected by seamless cloning, and then the connection products are transferred into WM3064 or DH5 alpha pir for overnight culture. After overnight culture on agar plates, a monoclonal colony was grown, and the colony was observed under a fluorescent microscope, and if the colony was green, it was preliminarily confirmed that pGGTOX plasmid construction was successful, and to further confirm that all the gene sequences had been successfully ligated to form a circular plasmid, we manually synthesized primers for the ligation interfaces of four gene sequences (tsPurpleF/GFP-AC-F; szu941-for/Apr-end; bsa-BsmB-F/pCasKp-id4386R; pKD46AntiF/Szu 941-rev) were subjected to PCR identification, and the identification results showed that the ligation interfaces of the 4 gene fragments all had bands (FIG. 5) the primer sequence was as follows with the construction pGGTOX plasmid system:

R6K-oriT-F:GGAGAAGTAATCGCTCACTGACCCGCTG;

R6K-oriT-R:TACCTTACTTTCTCATGAATCCCTTAACGTGAG;

gg-Apr-ggF:ATTCATGAGAAAGTAAGGTAAAGGCCTCGCG;

gg-Apr-ggR:TCGAGCGATATGGATCCCCAGCCTACACAA;

mqsR-F:TGGCCGCATGTGCCAGGCATCAAATAAAACGA;

mqsR-R:CAGTGAGCGATTACTTCTCCTTAAACGATACGATCAGTACG;

rhaS-GFP-F:ATTCATGAGAAAGTAAGGTAAAGGCCTCGCG;

rhaS-GFP-R:TCGAGCGATATGGATCCCCAGCCTACACAA;

tsPurpleF:CGCGACGGTTTCTTACAGTG;

GFP-AC-F:CAGAGTCGGCCAAGGGACCGGCAGTTTACC;

Szu941-for:GTAAGAGGATAACCCTGAGCTCGCTTGG;

Apr-end:CCGAGACACTGCACCATTCT;

Bsa-BsmB-F:AGTCTTGGTACGGAGCGAGA;

pCasKp-id4386R:GAGCGGGTGTTCCTTCTTCA;

pKD46AntiF:ACGAAAACTCACGTTAAGGGAT;

Szu941-rev:TTTACCTGGCCCAAATCGATAATGCTAGCA。

the seamless cloning kit was purchased from Yi mu (Beijing) technologies, inc., and the connection system was as follows:

recovery of products from multiple type II cleavage sites and APr resistant gene gels	1μL(40ng)
		SFGFP Gene fragment gel recovery product	1μL(50ng)
R6K gene fragment gel recovery product	2μL(46ng)
		MqsR gene fragment gel recovery product	0.7μL(50ng)
2×Seamless Cloning MIX	5μL
		dd H2O	1μL
Total volume of	10μL

Reaction at 50 ℃ 30s post reaction was transferred to WM 3064.

Constructing pCP-oriT plasmid, namely respectively amplifying the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene to obtain target gene fragment, and connecting the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene into a loop by using seamless cloning technology to obtain pCP-oriT plasmid.

Specifically, the procedure for constructing the pCP-oriT plasmid was similar to that of pGGTOX. We first cloned FLIP gene (FLP recombinase) and lambda-repressor gene (lambda-repressor) using the pair of primers 1088Rep-F and 1088Rep-R as templates, artificially synthesized primer pair 1088FLIP-F and 1088FLIP-R as well as chloramphenicol resistance gene and temperature sensitive replicon Rep101 gene fragment using plasmid pCP20 (construction process reference: one-step inactivation of chromosomal GENES IN ESCHERICHIA coli K-12using PCR products; literature website: https:// doi. Org/10.1073/pnas.120163297). Next, the pair of primers 1295hyg-F and 1295hyg-R was synthesized and the hygromycin resistance gene was amplified using plasmid pCasKp-hph (construction process reference ：CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae; literature website: https:// doi.org/10.1128/AEM.01834-18) as a template. Finally, the oriT fragment was amplified using primers 1211oriT-F and 1211oriT-R, and plasmid pSZU941-GFP as template. The four fragments are connected into a loop through a seamless cloning technology, and the construction of pCP-oriT is completed. The primer sequence and the seamless cloning system are as follows:

1088flip-F:GGGGCAAGGATCCTGGTATCGGTCTGCG;

1088flip-R:GCTTCTCACGGTTTGGTTGATGCGAGTG;

1088rep-F:CCAAACCGTGAGAAGCACACGGTCACAC;

1088rep-R:CCCGAAGCGCACCAAAAACTCGTAAAAGCT;

1295hyg-F:CAGCTAGCTGCAAACCCTCACTGATCCG;

1295hyg-R:CCAGGATCCTTGCCCCTCCAACGTCAT;

1211oriT-F:TTTGGTGCGCTTCGGGGTCATTATAGCGA;

1211oriT-R:GGTTTGCAGCTAGCTGTCAAGATATCAAGC。

the seamless cloning kit was purchased from Yi mu (Beijing) technologies, inc., and the connection system was as follows:

hygromycin gene gel recovery product	1μL(100ng)
		FLIP gene gel recovery product	1μL(50ng)
Chloramphenicol resistance gene recovery product	2μL(50ng)
		OriT gene fragment gel recovery product	0.7μL(150ng)
2×Seamless Cloning MIX	5μL
		dd H2O	1μL
Total volume of	10μL

Reaction at 50 ℃ 30s post reaction was transferred to WM 3064.

The nucleotide sequence of pGGTOX plasmid constructed above is shown as SEQ ID NO.2, and the nucleotide sequence of pCP-oriT plasmid is shown as SEQ ID NO. 3.

In still another embodiment of the present invention, there is provided a gene knockout method in enterobacteriaceae bacteria, the gene knockout method using the plasmid system of claim, comprising the steps of:

Determining gene fragments to be knocked out in enterobacteriaceae bacteria, amplifying two sides of a gene to be knocked out by designing a primer to obtain two homologous arm gene fragments, inserting the two homologous arm gene fragments into pGGTOX plasmids by a one-step method, converting the two homologous arm gene fragments into carrier bacteria WM3064 to serve as donor bacteria, taking enterobacteriaceae bacteria as acceptor bacteria, performing joint transfer on the donor bacteria and the acceptor bacteria, knocking out the gene fragments to be knocked out by utilizing a homologous recombination principle to obtain a recombinant strain, taking WM3064 converted with pCP-oriT plasmids as donor bacteria, performing joint transfer on the recombinant strain serving as acceptor bacteria, eliminating a resistance gene fragment in the middle of the homologous arm genes, and removing the pCP-oriT plasmids by increasing the culture temperature.

In still another embodiment of the present invention, there is provided a method for removing a drug-resistant plasmid from enterobacteriaceae bacteria, wherein the plasmid system removes the drug-resistant plasmid from enterobacteriaceae bacteria, comprising the steps of:

Determining a plasmid to be knocked out in enterobacteriaceae bacteria, amplifying a gene sequence in the plasmid to be knocked out by designing a primer to obtain two homologous arm gene fragments, inserting the two homologous arm gene fragments into pGGTOX plasmids by a one-step method, converting the two homologous arm gene fragments into carrier bacteria WM3064 to serve as donor bacteria, taking enterobacteriaceae bacteria as acceptor bacteria, performing conjugation and transfer on the donor bacteria and the acceptor bacteria, fusing pGGTOX plasmids with the plasmid to be knocked out by utilizing a homologous recombination principle to obtain a recombinant strain, and utilizing rhamnose to induce MqsR toxin gene expression to remove fusion plasmids, so that drug-resistant plasmids are removed.

In yet another embodiment of the present invention, there is provided the use of the above plasmid system for gene editing and drug resistant plasmid clearance in enterobacteriaceae including one or more of the genera escherichia, salmonella, shigella, klebsiella, serratia, yersinia, proteus, preferably enterobacteriaceae including escherichia and klebsiella.

Example 1

The DAPA gene sequence of Nissle1917 (purchased from Georgi, abbreviated as 1917) of Enterobacteriaceae was deleted using pGGTOX tool plasmid, comprising the steps of:

1. selection and acquisition of homology arms

The homologous arms of the gene clearing are taken as the homologous arms of 1000 bases close to the two sides of the DAPA motif, and the target fragment is amplified by PCR (polymerase chain reaction) of the homologous arms at the two sides by using artificial synthetic primers.

Name of the name	Sequence(s)
		PGG-BsaI-dap1F	GGCTACGGTCTCGGGATAGCTGCACTTTGTACGTCCA
PGG-BsaI-dap1R	GGCTACGGTCTCGGTGGCAATTAGGCGGTCATGGGGT
		PGG-BsaI-dap2F	GGCTACGGTCTCGGGAGCAAGCTAGCCCGACAGACAT
PGG-BsaI-dap2R	GGCTACGGTCTCGATGGCAGCACGGAATGGAGCAAAG

Two-sided homology arm PCR reaction system:

Left homologous arm (1000 bp)

Prime STAR Mix	25μL
		1917 Wild strain template	Diluting into bacterial liquid and adding 1 mu L
PGG-BsaI-dap1F	Final concentration 0.5. Mu.M
		PGG-BsaI-dap1R	Final concentration 0.5. Mu.M
dd H2O	Adjusting the volume of water according to the concentration of the template
		Total volume of	50μL

Right homologous arm (1260 bp)

Prime STAR Mix	25μL
		1917 Wild strain template	Diluting into bacterial liquid and adding 1 mu L
PGG-BsaI-dap2F	Final concentration 0.5. Mu.M
		PGG-BsaI-dap2R	Final concentration 0.5. Mu.M
dd H2O	Adjusting the volume of water according to the concentration of the template
		Total volume of	50μL

The reaction conditions are as follows:

The steps (1) at 95 ℃ for 5 minutes, (2) at 95 ℃ for 30 seconds, (3) at 55 ℃ for 30 seconds, (4) at 72 ℃ for 1 minute for 30 seconds, (5) at 72 ℃ for 30 seconds, and (2), (3) and (4) are repeated 35 times.

2. Construction of tool plasmid pGGTOX-dap with homology arm of target gene by one-step method

The gene sequence in step 1 was subjected to agarose gel recovery, and the concentration of the left homology arm was 50 ng/. Mu.L, the concentration of the right homology arm was 36 ng/. Mu.L, and the concentration of pGGTOX plasmid (5471 bp) was 50 ng/. Mu.L. Thereafter, the ligation was performed with pGGTOX. The homologous arm fragment of the DAPA gene sequence was inserted into the pGGTOX plasmid by BsaI cleavage and T4 ligation in conjunction with the Golden Gate cloning procedure.

Golden Gate clone reaction system:

The reaction conditions are as follows:

(1) 37 ℃ for 20 minutes, (2) 37 ℃ for 5 minutes, (3) 37 ℃ for 5 minutes, and (2) and (3) repeating the steps for 50 cycles.

3. Making it enter carrier bacteria by chemical/electric conversion

We electrotransformed or chemotransformed the constructed tool plasmid into WM3064 using WM3064 (E.coli WM3064, purchased from NTCC, supra) as a carrier strain. The cells were plated with 100. Mu.L of the bacterial liquid overnight.

Identification of pGGTOX-dap

The phenotype can be identified by a fluorescence microscope, and if the strain has obvious green fluorescence under the microscope, the pGGTOX-dap tool plasmid construction can be initially proved to be successful in entering the carrier strain WM 3064. This strain was then enriched and kept in a pool and allowed to undergo conjugal transfer as donor strain.

5. Preparation of recipient strains

The pKD46 (purchased from Ubbelopsis) plasmid was electrotransferred or chemotransferred into 1917 using wild-type Nissle1917 as a vector strain. The cells were plated with 100. Mu.L of the bacterial liquid overnight. The successfully transformed strain is selected for storage and allowed to undergo conjugal transfer as a recipient strain.

6. Conjugation transfer with Nissle1917 Using homologous recombination principles to knock out DAPA Gene sequence

Conjugation transfer was performed using WM3064 with pGGTOX-dap transfer as donor and 1917-pKD46 prepared in 5 as acceptor, and the donor was dropped onto LB agar plates with DAPA plus 2% glucose at a ratio of 2:1, and incubated overnight. The pGGTOX-dap plasmid was transferred into 1917-pKD46 and the zygotes were screened the next day using Apr plus 2% GLU agar plates. The DAPA gene is knocked out by using a homologous recombination method. The observation of the strain as green proves that the homology arm side of pGGTOX-dap plasmid is integrated with 1917-pKD46, and the homologous recombination is needed for the second induction.

7. And (3) inducing to carry out secondary homologous recombination.

According to 6, pGGTOX-dap after the first homologous recombination on the side homology arm was combined with 1917-pKD46, we induced a second homologous recombination by adding Apr and DAPA and 2% glucose in LB broth at 37℃and shaking the bacterial suspension to OD 0.5-0.6. After re-suspending the bacterial suspension twice with a mixture of 2% mouse Li Tangjia M9, 100. Mu.L of the bacterial suspension was applied to M9 plus Apr plus DAPA plates and plates with only Apr and M9, and the plates were diluted the next day after half of the bacterial suspension was left overnight in the refrigerator to prevent the second day from excessive bacterial load on M9 agar plates and failing to screen out single colonies. After the end of the secondary homologous recombination, 2 cases will occur, the first being the strain from which the gene of interest was successfully knocked out, and the second being the strain which was restored to the wild strain Nissle1917 after the secondary homologous recombination. We therefore performed a preliminary phenotypic screen of the target strain with DAPA and M9 agar plates without DAPA.

8. Whether the target gene is knocked out is identified, and feasibility of pGGTOX plasmids is confirmed.

After overnight incubation after the first plating, no single clone was separated and no significant difference in the amounts of DAPA was observed between the DAPA and the agar plates without DAPA, we diluted the stock in the refrigerator ten times and plated 100ul again, and the next day we found an interesting phenomenon in which the stock in the refrigerator overnight grew in a very large amount on the DAPA plates and hardly on the plates without DAPA (FIG. 6A). The bacterial metabolism is slowed down probably because of the low temperature environment, and the pGGTOX-dap plasmid is easier to carry out secondary homologous recombination on the DAPA gene sequence of the Nissle1917 wild strain under the action of exogenous supplement DAPA, so that the DAPA gene sequence is replaced. The working efficiency is obviously higher than that of the refrigerator without being put. The successful knockout of the DAPA gene sequence was confirmed after identification of the interface on both sides of the homology arms and phenotypic identification by means of synthetic primers (dap-ext-R/ggtox-R-esaF; dap-ext-F/ggtox-R-esaRPCR) (FIG. 6B). We used this method to knock out the target gene from the plasmid of the work pGGTOX, which demonstrates the feasibility of the tool plasmid. The primer sequences are as follows:

dap-ext-R:GCAGGGCAGTGAGAAGATTTG;ggtox-R-esaF:CATGAGCTCACTAGTGGATCCC;

dap-cxt-F:GCTGTCGGTCACTTTCATGC;ggtox-R-esaR:GCCTCGAGAATTCTGACGTCT。

9. Elimination of resistance gene by pCP-oriT plasmid and verification

The modified plasmid pCP-oriT encoding FRT site-specific FLP recombinase is subjected to joint transfer with the recombinant strain identified in the experiment, the recombinant strain is subjected to inversion culture at the temperature of 30 ℃ in a ratio of 4:1, a loop of fungus membrane is taken out on a solid culture set with chloramphenicol by a fungus taking loop for the next day, the recombinant strain is subjected to inversion overnight culture at the temperature of 30 ℃ to obtain positive binders, the Flip recombinase is induced to act at the same time, then a plurality of monoclonal clones are selected for PCR verification or phenotype verification to verify whether the Apr resistance gene exists, the identified monoclonal clones with the Apr resistance of negative are selected for transferring to LB culture medium, the culture is carried out for 12 hours at the temperature of 37 ℃, and the pCP-oriT plasmid is removed (FIG. 9).

Example 2

Method for removing mrk plasmid in enterobacteria by using pGGTOX tool plasmid

1. Design and acquisition of homology arms

700 Bases were selected as homology arms on one side of the mrk plasmid mrk gene sequence. The target fragment was amplified by PCR using artificial primers for both homology arms.

Name of the name	Sequence(s)
		pGG-rm-mrk2R-P	GGCTACCGTCTCAATGGCGCCGGGACAATTTTATTACTCT
pGG-rm-mrk2F-P	GGCTACCGTCTCAGGAGCACTACCGCTACCATGGGC
		pGG-rm-mrk1R-P	GGCTACCGTCTCAGTGGCAGATAGGCGCTGCTGTTAT
pGG-rm-mrk1F-P	GGCTACCGTCTCAGGATGTAAAACCGTGATGCTGGCG

PCR reaction system with two homology arms

Left homologous arm (700 bp)

Prime STAR Mix	25μL
		Mrk plasmid as template	Diluted to 1ng plus 1. Mu.L
pGG-rm-mrk1R-P	Final concentration 0.5. Mu.M
		pGG-rm-mrk1F-P	Final concentration 0.5. Mu.M
dd H2O	Adjusting the volume of water according to the concentration of the template
		Total volume of	50μL

Right homologous arm (700 bp)

2. Construction of tool plasmid pGGTOX-mrk with homology arm of target gene by one-step method

The gene sequence in step 1 was subjected to agarose gel recovery, and the concentration of the left homology arm was 128 ng/. Mu.L, the concentration of the right homology arm was 69 ng/. Mu.L, and the concentration of pGGTOX plasmid (5471 bp) was 140 ng/. Mu.L. Then ligated pGGTOX by inserting the homologous arm fragment of the DAPA gene sequence into the pGGTOX plasmid by cleavage, T4 ligation and Golden Gate cloning.

The Golden Gate clone reaction system.

PGGTOX plasmid	420ng
		Left homologous arm segment	25.6ng
Right homology arm fragment	23ng
		NEB Golden Gate Mix	1μL
Buffer T4 DNA Ligase	2μL(10X)
		Nuclease-free Water	To20μL

The reaction conditions are as follows:

(1) 42 ℃ for 2 minutes, (2) 16 ℃ for 2 minutes, (3) 60 ℃ for 2 minutes, (1) and (2) repeating the steps for 60 cycles.

3. Making it enter carrier bacteria by chemical/electric conversion

We used WM3064 as the vector strain to electrotransduce or transduce the constructed tool plasmid into WM 3064. The cells were plated with 100. Mu.L of the bacterial liquid overnight.

Identification of pGGTOX-mrk

The phenotype can be identified by a fluorescence microscope, and if the strain has obvious green fluorescence under the microscope, the construction of pGGTOX-mrk tool plasmids and the successful entry into the carrier bacteria WM3064 can be initially demonstrated. The strain is then enriched and kept in a bacterial pool.

5. Conjugation transfer with clinical Strain harboring mrk plasmid mrk plasmid was knocked out Using homologous recombination principle

The conjugation transfer was performed using WM3064 transferred to pGGTOX-mrk as donor and clinical strain JNQH950 carrying mrk plasmid (strain bioinformatics has been uploaded to NCBI, bioSample: SAMN36745160; SAMPLE NAME: JNQH950; currently stored in the Shandong first medical university affiliated Hospital (Qianfhan Hospital)) as recipient, and was incubated overnight on LB agar plates with DAPA plus 2% glucose at a donor to recipient ratio of 1:1. The pGGTOX-mrk plasmid was transferred into the clinical strain JNQH950 carrying the mrk plasmid, and the mrk plasmid was knocked out by homologous recombination. The zygote phenotype was observed under a fluorescence microscope and if the zygote was observed to be green under the mirror, it was confirmed that the pGGTOX-mrk plasmid had been successfully inserted into the mrk plasmid in its entirety by one homologous recombination.

6. The plasmid mrk was cleared by rhamnose induced expression of the MqsR toxin gene.

According to the description 5, pGGTOX-mrk, after the first homologous recombination, the whole plasmid was combined with mrk plasmid, and the mrk plasmid was knocked out together with pGGTOX tool plasmid by inducing the MqsR gene expression downstream of the promoter by rhamnose, and the bacterial solution was shaken to OD value of 0.5-0.6 by inducing in LB broth added with 2% glucose. After re-suspending the bacterial suspension twice with a 2% mouse Li Tangjia M9 mixture, 100. Mu.L of the bacterial suspension was applied to the M9 plates and the plates with Apr and M9 were incubated overnight, since we performed a preliminary phenotypic screen of the target strain with Apr and M9 agar plates without Apr.

7. Whether the mrk plasmid is knocked out is identified, and the feasibility of eliminating the enterobacteria drug-resistant plasmid by pGGTOX is confirmed.

After overnight culture, monoclonal colonies are grown on the agar plates, ten monoclonal colony synthetic primers Incx F/Incx R and mrkA-1F/mrk-locL 236 are selected to identify the homology arm interface and fragments and replicons on mrk plasmid, and the result shows that only the fourth strain of single colony in ten strains has no band, which proves that the fourth strain loses mrk plasmid. As a result, as shown in FIG. 7, the plasmid of the functional pGGTOX was knocked out by using this method, and the feasibility of the plasmid was confirmed. The primer sequences are shown below:

Incx1F：TCCAGGTTGCCGTAATCAC;Incx1R：ATGGGCTGTATTCTGGCTGG;mrkA-1F：GGTAAACGTTCGCATCGCTG;mrk-locL：GCATTCTTTGACGCCGATAGC.

8. Elimination of resistance gene by pCP-oriT plasmid and verification

As in step 9 of example 1.

Example 3

The TA gene sequence of Klebsiella pneumoniae is cleared by utilizing pGGTOX tool plasmid, the TA gene sequence is often found in Salmonella, but we laboratory found a Klebsiella pneumoniae carrying TA gene, which encodes its triple flagella gene, we input the strain into a bacterial pool named JNQH327 (biological sample information of the strain has been uploaded to NCBI, bioSample: SAMN43766382, SAMPLE NAME: JNQH327; currently stored in Shandong university of first medical science affiliated Hospital (Qianshan Hospital)), and then we use pGGTOX tool plasmid to knock out the TA gene and observe the phenotype and change after the strain loses the TA gene sequence, comprising the following steps:

1. selection and acquisition of homology arms

The target fragment is amplified by PCR with artificial synthetic primer and the two homologous arms with 700 bases of the common sequence near the two sides of TA motif as homologous arms for gene elimination.

PCR reaction system with two homology arms

Left homologous arm (700 bp)

Prime STAR Mix	25μL
		JNQH327 strain template	Diluting into bacterial liquid and adding 1 mu L
pGG-vap1F	Final concentration 0.5. Mu.M
		pGG-Vap1R	Final concentration 0.5. Mu.M
dd H2O	Adjusting the volume of water according to the concentration of the template
		Total volume of	50μL

Right homologous arm (700 bp)

Prime STAR Mix	25μL
		JNQH327 strain template	Diluting into bacterial liquid and adding 1 mu L
pGG-Vap2F	Final concentration 0.5. Mu.M
		pGG-Vap2R	Final concentration 0.5. Mu.M
dd H2O	Adjusting the volume of water according to the concentration of the template
		Total volume of	50μL

The reaction conditions are as follows:

Pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 40 sec, final extension at 72 ℃ for 30 sec, denaturation, annealing and extension steps were repeated 35 times.

2. Construction of tool plasmid pGGTOX-TA with homologous arm of target gene by one-step method

The gene sequence in step 1 was subjected to agarose gel recovery, and the concentration of the left homology arm was 58 ng/. Mu.L, the concentration of the right homology arm was 33 ng/. Mu.L, and the concentration of pGGTOX plasmid (5471 bp) was 50 ng/. Mu.L. Then ligated pGGTOX by inserting the homologous arm fragment of the DAPA gene sequence into the pGGTOX plasmid by cleavage, T4 ligation and Golden Gate cloning.

Golden Gate clone reaction system:

pGGTOX plasmid	100ng
		Left homologous arm segment	26ng
Right homology arm fragment	26ng
		NEB Golden Gate Mix	1μL
Buffer T4 DNA Ligase	2μL(10X)
		Nuclease-free Water	To20μL

The reaction conditions are as follows:

(1) 42 ℃ for 2 minutes, (2) 16 ℃ for 2 minutes, (3) 60 ℃ for 2 minutes, (1) and (2) repeating the steps for 60 cycles.

3. Making it enter carrier bacteria by chemical/electric conversion

We used WM3064 as the vector strain to electrotransduce or transduce the constructed tool plasmid into WM 3064. The cells were plated with 100. Mu.L of the bacterial liquid overnight.

Identification of pGGTOX-TA

The phenotype can be identified by a fluorescence microscope, and if the strain has obvious green fluorescence under the microscope, the pGGTOX-TA tool plasmid construction can be initially proved to be successful in entering the carrier strain WM 3064. The strain is then enriched and kept in a bacterial pool.

5. Conjugation transfer with klebsiella pneumoniae carrying TA gene sequence and knockout of TA gene sequence by homologous recombination principle

The recipient JNQH strain 327 was subjected to conjugal transfer using WM3064 transferred to pGGTOX-TA as donor, and incubated overnight on LB agar plates with DAPA plus 2% glucose at a donor to recipient ratio of 3:1. The pGGTOX-TA plasmid is transferred into JNQH strain 327, and the TA gene sequence is knocked out by using a homologous recombination method. The phenotype is observed under a fluorescence microscope, and when the strain is green, the fact that the pGGTOX-TA plasmid homology arm side is combined with JNQH327 strain into a whole is proved, and the homologous recombination is needed to be performed through a second induction.

6. And (3) inducing to carry out secondary homologous recombination.

According to the description 5, pGGTOX-TA after the first homologous recombination, one side homology arm and JNQH strain 327 were combined together, we induced the second homologous recombination, we induced with LB broth added with Apr and 2% glucose, and the bacterial solution was shaken to OD 0.5-0.6. The bacterial suspension was resuspended twice in a mixture of 2% murine Li Tangjia M9, and in view of the experience given in example 1, we placed 100. Mu.L of the bacterial suspension in a4℃refrigerator overnight and spread on plates of Apr and M9.

7. Whether the target gene is knocked out is identified, and feasibility of pGGTOX plasmids is confirmed.

After the end of the secondary homologous recombination, 2 cases will occur, the first being the strain from which the gene of interest was successfully knocked out, and the second being the strain which was restored to JNQH wild-type strain after the secondary homologous recombination. The next day, a plurality of monoclonal colonies were grown on the plate, 16 strains of monoclonal were selected and PCR identification was performed on the TA gene sequence by using the synthetic primer COG-idF/COG-idR, and the result shows that half of the monoclonal colonies have been successfully knocked out the TA gene sequence as shown in FIG. 8. The primer sequences are as follows:

COG-idF:TCATACGCTCCTGTCTCGGA;COG-idR:ATCAATGAAGGCCTGGACGG。

The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. A plasmid system for traceless gene editing and drug resistant plasmid clearing in enterobacteriaceae bacteria is characterized by comprising pGGTOX plasmids with gene editing and plasmid clearing and a pCP-oriT plasmid with resistance gene elimination, wherein pGGTOX comprises mqsR genes, R6K replicons, rhaS genes, SFGFP genes and Apr genes which are connected in sequence, and pCP-oriT comprises FLIP genes, lambda-repressor genes, cmR genes, rep101 genes, hyg genes and oriT genes which are connected in sequence.

2. The plasmid system of claim 1 wherein the Enterobacteriaceae bacteria include one or more of the genera Escherichia, salmonella, shigella, klebsiella, serratia, yersinia, and Proteus, preferably, the method comprises the steps of, the Enterobacteriaceae bacteria include the genus Escherichia and the genus Klebsiella.

3. A method of constructing a plasmid system according to any one of claims 1-2, characterized by comprising the steps of:

Constructing pGGTOX plasmids, namely respectively amplifying an R6K gene, an Apr gene, an MqsR gene, a rhaS gene and a SFGFP gene to obtain target gene fragments, using PGGASELSCT plasmids as templates, amplifying the amplified large gene fragments by using primers pGGE1 and pGGE2, carrying out phosphorylation and cyclization by using T4 enzyme to obtain pGGA plasmids, inserting the Apr gene into the pGGA plasmids to obtain pGGB plasmids, amplifying the Apr gene with enzyme cutting sites on two sides by using the primers, and connecting the R6K gene, the Apr gene with enzyme cutting sites on two sides, the MqsR gene, the rhaS gene and the SFGFP gene fragments by a one-step method to obtain pGGTOX plasmids;

Constructing pCP-oriT plasmid, namely respectively amplifying the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene to obtain target gene fragment, and connecting the IP gene, lambda-repressor gene, cmR gene, rep101 gene, hyg gene and oriT gene into a loop by using seamless cloning technology to obtain pCP-oriT plasmid.

4. The method of claim 3, wherein in the step of constructing pGGTOX plasmids, two pairs of BsmBI and BsaI cleavage sites are introduced flanking the Apr gene.

5. A method of gene knockout in enterobacteriaceae, characterized in that the plasmid system of any one of claims 1-2 is used for gene knockout.

6. The gene knockout method of claim 7, comprising the steps of:

Determining gene fragments to be knocked out in enterobacteriaceae bacteria, and amplifying two sides of the gene to be knocked out by designing primers to obtain two homologous arm gene fragments;

Inserting the two homologous arm gene fragments into pGGTOX plasmid by a one-step method, converting into carrier bacteria WM3064 to serve as donor bacteria, and taking enterobacteriaceae bacteria as acceptor bacteria;

Performing joint transfer on the donor bacteria and the receptor bacteria, and knocking out a gene fragment to be knocked out by utilizing a homologous recombination principle to obtain a recombinant strain;

WM3064 transformed with pCP-oriT plasmid was used as donor strain, the recombinant strain was used as acceptor strain, conjugation transfer was performed to eliminate the resistance gene fragment with homologous arm gene in between, and pCP-oriT plasmid was removed by increasing the culture temperature.

7. A method for clearing a drug-resistant plasmid in enterobacteriaceae bacteria, characterized in that the plasmid system according to any one of claims 1-2 is used for clearing a drug-resistant plasmid in enterobacteriaceae bacteria.

8. The method for removing drug-resistant plasmid from enterobacteria of claim 7, comprising the steps of:

determining a plasmid to be knocked out in enterobacteriaceae bacteria, and amplifying a gene sequence in the plasmid to be knocked out by designing a primer to obtain two homologous arm gene fragments;

Inserting the two homologous arm gene fragments into pGGTOX plasmid by a one-step method, converting into carrier bacteria WM3064 to serve as donor bacteria, and taking enterobacteriaceae bacteria as acceptor bacteria;

And performing conjugation transfer on the donor bacteria and the receptor bacteria, fusing pGGTOX plasmids with plasmids to be knocked out by utilizing a homologous recombination principle to obtain recombinant strains, and utilizing rhamnose to induce MqsR toxin gene expression to remove fusion plasmids, so that drug-resistant plasmids are removed.

9. Use of the plasmid system of claim 1 for gene editing and drug resistant plasmid clearance in enterobacteriaceae bacteria.

10. The use according to claim 9, wherein the Enterobacteriaceae family comprises one or more of the genera Escherichia, salmonella, shigella, klebsiella, serratia, yersinia, proteus, preferably, the method comprises the steps of, the enterobacteriaceae include the genera escherichia and klebsiella.