Vibrio mimicus TA system toxin protein, encoding gene and application thereof

Abstract

The invention discloses a vibrio mimicus TA system toxin protein, an encoding gene and an application thereof. An amino acid sequence of the vibrio mimicus TA system toxin protein is represented as the SEQ ID No.2 and the amino acid sequence of the encoding gene of the vibrio mimicus TA system toxin protein is represented as the SEQ ID No.1. In the invention, for the first time, the TA system is found in the vibrio mimicus, and a key toxin gene vmi480 in the TA system is proved to be a novel toxin gene, of which a product has a strong lethal effect on bacterial cells with a wide toxicity effect range, so that the vmi480 can be used for replacing lethal genes, such as ccdB, sacB and the like, which are widely applied but are serious in false positive. The vmi 480, when being used in construction of cloning vectors and suicide vectors, can greatly increase screening efficiency of positive recons, thereby reducing workload in laboratory.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a Vibrio mimicus TA system toxin protein, its coding gene and its application. Background technique: [0002] The toxin-antitoxin (TA) system was originally discovered in some low-copy plasmids. They are a pair of closely related gene combinations, one of which encodes a stable toxin and the other encodes a less stable antitoxin (Wang Xiaolei et al., 2008 ). When a bacterial cell divides, if the progeny cells lose the plasmid, the less stable antitoxin will degrade quickly, while the stable toxin will exert toxicity to kill the cell. This effect is called post-division lethal effect, and the plasmid can be released Stable in the host (Jump et al., 2001). With the development of biological sequencing technology and bioinformatics analysis technology, 671 TA system loci have been found in sequenced prokaryotes by 2008, distributed in 126 species, and they...

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Problems solved by technology

However, in practical applications, we found that E. coli containing ccdB-derived plasmids still had some colonies on the plate under the induction of IPTG, indicating that the toxicity of ccdB was not strong enough to kill all E. coli cells or bacterial cells It is easy to self-modify the target site of ccdB; in addition, it is also found that when the ccdB-derived suicide vector undergoes the first homologous recombination and integrates into the Vibrio genome, it is completely unable to kill Vibrio cells under the induction of IPTG , which indicates that the toxicity of ccdB expression products is host-specific

The expression of the sacB lethal gene derived from Bacillus metabolizes sucrose and produces metabolites that are lethal to most Gram-negative bacteria (Gay et al., 1985), however, it was later found that in homologous recombination based on the sacB suicide plasmid, some bacteria were Sucrose metabolites are not sensitive, so the screening of deletion mutants is not easy to succeed (Milton et al., 1996); in addition, sacB is prone to spontaneous point mutations, resulting in a large number of false positive clones during screening (Blomfield et al., 2006)

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