Construction method of vibrio parahaemolyticus gene efficient knockout plasmid

Abstract

The invention discloses a construction method of a vibrio parahaemolyticus gene efficient knockout plasmid, and belongs to the field of molecular biology and biotechnology. According to the method, the knockout process is optimized, and a Cre / loxP system is introduced. A gentamicin resistant fragment with a loxP site is added between knockout homologous arms, and then the resistant fragment is removed through Cre enzyme. Gentamicin is added during the second homologous recombination, and only the strains which are correctly subjected to homologous recombination can survive on a 10% sucrose gentamicin resistance plate, so that the screening efficiency of the knockout strains is improved. And carrying out second binding transduction, and introducing the pOTC plasmid with the cre gene into the vibrio parahaemolyticus. The cre gene expresses Cre enzyme in cells, recognizes loxP sites, removes gene segments between loxL and loxR, that is, removes gentamicin resistant segments, and removes pOTC plasmids in a test tube containing 10% of sucrose, and the method improves the screening efficiency of correct strains.

Description

technical field [0001] The invention relates to a method for constructing a high-efficiency gene knockout plasmid of Vibrio parahaemolyticus, belonging to the fields of molecular biology and biotechnology. Background technique [0002] The traditional knockout process of Vibrio parahaemolyticus is as follows: the homology arm is connected to the suicide plasmid, transduced into the cell by conjugation and integrated into the genome by homologous recombination. Secondly, in the environment of 10% sucrose, the sacB integrated into the genome plasmid will decompose the sucrose to produce lethal substances, thus forcing the cells to undergo the second homologous recombination to remove the plasmid. There are two situations in the second homologous recombination. The first one is that the location of homologous recombination is different from that of the first step, that is, the homologous recombination of the first step and the second step occur on the upstream / downstream homolo...

AI Technical Summary

Problems solved by technology

There will be two situations in the second homologous recombination. The first one is that the location of homologous recombination is different from that of the first step, that is, the homologous recombination of the first step and the second step occur on the upstream / downstream homology arms respectively. , so that when the plasmid is removed, the target gene fragment will also be removed, and the knockout is successful; the second case is that the homologous recombination occurs at the same position as the first step, that is, the homologous recombination between the first step and the second step Occurs on the same homology arm, and the result is that the plasmid integrated on the genome is removed intact, and the knockout fails

In the knockout process of some genes, the second step can consume a lot of time and resources

[0003] Moreover, the sacB gene is prone to mutations, and its reverse screening has a high false positive rate, so it is necessary to identify a large number of mutant strains in the end, which takes a lot of time and resources

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