Expression cassette mazF-cassette for expressing escherichia coli toxin protein mazF gene and construction method and application thereof

Abstract

The invention discloses an expression cassette mazF-cassette for expressing escherichia coli toxin protein mazF gene, the nucleotide sequence of which is SEQ ID NO:1. The invention also discloses a construction method of the expression cassette mazF-cassette, comprising the following steps of: 1) amplifying the escherichia coli toxin protein mazF gene; 2) amplifying xylose-induced promoter Pxyl A gene; 3) amplifying a resistant gene zeocin; 4) fusing the above three PCR segments into a recombinant PCR segment; 5) carrying out enzyme digestion on the recombinant PCR segment by the use of restriction enzyme Sal I / Xba I, connecting with an integrative vector Psg1729 which undergoes the same enzyme digestion, transforming bacillus subtilis 1A751 to obtain a transformant which is a host bacteria containing the expression cassette. The expression cassette mazF-cassette can be used to construct a bacillus subtilis general label-free homologous recombination system.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for site-directed mutation, deletion and introduction of exogenous genes. Background technique [0002] Bacillus subtilis is a type of aerobic rod-shaped bacteria. Under extreme conditions, it can also induce the production of endogenous spores with strong stress resistance. It widely exists in soil, lakes, oceans, etc., and has no pathogenicity itself. It has only a single layer of outer membrane and can directly secrete many proteins into the medium. Bacillus subtilis is the first genetically engineered expression strain in Bacillus, and it is widely used in the production of industrial enzymes. Therefore, B. subtilis has received more and more attention as an expression strain. [0003] The vectors of Bacillus subtilis expression system mainly include self-replicating plasmids, integrating plasmids and phages. According to the different replication metho...

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Problems solved by technology

However, the presence of marker genes in modified bacterial strains has brought a lot of inconvenience to the follow-up research of bacterial strains, and has the following disadvantages: one, the marker genes that can be used as Bacillus subtilis screening genes for genetic manipulation are very limited at present, when a certain bacterial strain has been When there is a selectable marker gene, the marker gene can no longer be used as a selectable marker in the subsequent genetic manipulation, and the antibiotic gene must be deleted or replaced by other resistance genes; two, introducing too many antibiotic resistance genes in a bacterial strain often It will have adverse effects on some physiological states of the strain, such as affecting the expression of adjacent genes, etc.

However, this system also has some shortcomings, such as it requires restriction endonuclease digestion and ligation process, and the cycle is long, and it takes about 2 weeks to construct a recombinant; Leaky expression leads to natural mutations to produce mazF-resistant strains; recently, different scholars have improved the system (Morimoto et al. The cycle and leakage of toxin proteins have been improved, but there are still big problems. The deletion or insertion inactivation of genes requires at least 3600bp recombinant PCR fragments, and still requires restriction endonuclease treatment and ligation processes. Long-segment PCR products It is bound to affect the amplification efficiency and accuracy of PCR

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