Universal gene-knockout suicide vector for vibrios and application thereof

A gene knockout and suicide carrier technology, applied in the field of microbial genetic manipulation, can solve the problems of low reverse selection plate ratio, deletion mutation screening, time-consuming and labor-intensive work, etc., to increase the frequency of conjugation transfer and the probability of correct integration, lethal The target is wide, the effect of the rate improvement

Inactive Publication Date: 2015-11-18
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene knockout technology that can be successfully used for homologous recombination in bacteria such as Escherichia coli has some obvious defects in Vibrio gene knockout, including: (1) lack of versatility, limited to gene deletion in a certain Vibrio; (2) sacB is widely used as a lethal gene for suicide vectors, but the lethal effect induced by sacB is very weak in Vibrio, resulting in a very low proportion of correct deletion mutant clones in the reverse selection plate, resulting in a large number of deletion mutations Screening work; (3) It is time-consuming and labor-intensive to perform double enzyme digestion on the overlapping PCR product of the target fragment and the suicide plasmid

Method used

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  • Universal gene-knockout suicide vector for vibrios and application thereof
  • Universal gene-knockout suicide vector for vibrios and application thereof
  • Universal gene-knockout suicide vector for vibrios and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The construction of the universal gene knockout suicide vector pLP12T of embodiment 1 Vibrio (flow process is as follows figure 2 shown)

[0033] (1) Using the plasmid pSW23T (provided by Vincent Burrus, source literature DemarreG, etal. Research in Microbiology, 2005, 156:245–255) as a template, PCR amplification was performed using primers pSW23T-F / pSW23T-R, and the polymerase was TaKaRa company high-fidelity DNA polymerase PrimeSTAR (unless otherwise specified, all polymerases used in the present invention are this enzyme). Refer to the enzyme manual for PCR amplification conditions. Obtain PCR product, this PCR product comprises oriV R6Kγ , oriT RP4 , cat, fragment of the MCS site.

[0034] (2) Using pSW25T-ccdB (provided by Vincent Burrus, source http: / / openwetware.org / wiki / Mazel / PSW25T-ccdB.docx) as a template, PCR amplification was performed using primers pSW25T-F / pSW25T-R. A PCR product is obtained, which contains P TAC Promoter, ccdB, and a fragment cont...

Embodiment 2

[0041] Example 2 Application of pLP12T to knock out the Vibrio alginolyticus hemolysin gene (vah)

[0042] (1) The in-frame deletion fusion fragment of the vah gene of Vibrio alginolyticus E0601 was amplified by overlapping PCR. The first round of PCR primers were vah-MF1 / vah-MR1, vah-MF2 / vah-MR2, respectively, and the template was Vibrio alginolyticus E0601 genomic DNA. The first round produced PCR products vah1, vah2, respectively. After vah1 and vah2 were purified by PCR product purification kit (Axygen), equal volumes were mixed as templates for the second round of PCR. The second round of PCR uses primers vah-MF1 / vah-MR2, common TaqDNA polymerase (TaKaRa), and PCR amplification conditions refer to the enzyme instructions for PCR amplification conditions, thereby obtaining the second round of PCR products.

[0043] (2) After the second-round PCR product is purified, it is directly connected to the gene knockout suicide vector pLP12T commonly used in Vibrio, using T4 DNA ...

Embodiment 3

[0049] Example 3 Knockout of Vibrio cholerae periplasmic serine peptidase gene (degS) using pLP12T

[0050] (1) An in-frame deletion fusion fragment of the degS gene of Vibrio cholerae HN375 was amplified by overlapping PCR. The first round of PCR primers were degs-MF1 / degs-MR1, degs-MF2 / degs-MR2, and the template was Vibrio cholerae HN375 genomic DNA. The two PCR products were mixed in equal volumes and purified to serve as templates for the second round of PCR. The second round of PCR uses primers degs-MF1 / degs-MR2 and ordinary TaqDNA polymerase to obtain the second round of PCR products.

[0051] (2) The above-mentioned second-round PCR product is directly connected with the suicide vector pLP12T. The ligation product (plasmid pLP12-degS) was transformed into Escherichia coli DH5αλpir cells, and spread on LB plate (containing 20 μg / ml chloramphenicol, 0.3% D-glucose). Use degs-MF1 / degs-MR2 primers to perform PCR screening on the clones on the plate, and the correct clone...

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Abstract

The invention discloses a universal gene-knockout suicide vector for vibrios and a construction method theroef and provides an application thereof in gene knockout of the vibrios. The universal gene-knockout suicide vector pLP12 is a ring-shaped vector and comprises a PBAD promoter, a repressor protein gene araC, an RP4 transferring initiation site, a chlorampenicol resistant gene, an R6K duplicating initiation site, a multiple-cloning-site area and a lethal gene vmi480; the multiple-cloning-site area at least contains two AhdI restriction enzyme digestion sites; the suicide vector pLP12 is subject to AhdI restriction enzyme digestion to form linearized suicide vector pLP12T. The universal gene-knockout suicide vector adopts entirely-new reverse selection genes vmi480 and is used for replacing the common sacB gene. Foreign fragments carried by the pLP12T are transferred to vibrio cells to be mutated by a jointing mode, under the pressure of antibiotics and reverse selection of products of lethal gene vmi480, first-time homologous recombination and second-time homologous recombination are carried out on the vibrios successively, and finally the mutant strain with deletion of target genes is generated.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic manipulation, and in particular relates to a universal gene knockout suicide vector for Vibrio and its application. Background technique: [0002] Vibrio is the most common heterotrophic bacteria in the marine environment. At least 12 species of Vibrio can cause human disease, among which Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus are the most serious hazards to human health. In addition, many types of Vibrio are also pathogens of aquatic economic animals, and the vibriosis caused by them leads to a large amount of economic losses in aquaculture. Although some key pathogenic genes of Vibrio cholerae and Vibrio parahaemolyticus have been discovered very early, scientists' understanding of the pathogenic mechanism of Vibrio still lags behind the pathogenic bacteria of Enterobacteriaceae. The lack of mature and convenient genetic manipulation technology is the main obstacle for...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/03C12N1/20
CPCC12N1/20C12N15/03C12N15/63C12R2001/63C12N1/205
Inventor 罗鹏何香燕刘秋婷胡超群
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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