Method for improving stability of recombinant plasmid by chemical-mutagenesis saccharomyces cerevisiae genetically engineered bacterium

Abstract

The invention discloses a method for improving stability of recombinant plasmid by chemical-mutagenesis saccharomyces cerevisiae genetically engineered bacterium. The method comprises the following steps: (1), chemical mutagenesis; (2), screening; (3), detecting plasmid stability by PCR (Polymerase Chain Reaction); (4), induced expression and activity detection. According to the method, a saccharomyces cerevisiae gene engineering strain is mutated by chemical mutagendiethyl sulfate, and a culture medium containing pentafluorouracil is used for screening, so that cells capable of utilizing the uracil in the culture medium are sifted out to obtain a mutant incapable of utilizing the uracil in the culture medium. The mutant only can or almost only can use the plasmid gene to synthesize the uracil, and therefore, the stability of the plasmid is still kept in a non-selective culture medium, cost of the culture medium is greatly lowered, and one of key difficult problems that an antibacterial peptide is produced on a large scale by utilizing saccharomyces cerevisiae genetically engineered bacterium is restricted is overcome.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for chemically inducing Saccharomyces cerevisiae genetically engineered bacteria to improve the stability of recombinant plasmids. Background technique [0002] Antimicrobial peptides are small molecular active peptides produced by the immune defense system of organisms under inducing conditions, and are widely distributed in plants, animals and humans. Antimicrobial peptides are encoded by genes and synthesized by ribosomes. At present, more than one thousand antimicrobial peptides have been discovered and identified, most of which have a net positive charge. From the structural point of view, it can be divided into α-helical type, β-sheet type, rich in disulfide bonds, rich in proline and other types. Antimicrobial peptides have a broad antibacterial spectrum, and some antimicrobial peptides can also specifically inhibit the growth of certain tumor...

AI Technical Summary

Problems solved by technology

However, the selective medium is a synthetic medium, which requires the addition of some amino acids and non-basic nitrogen sources, and the cost is very high, which constitutes one of the main obstacles to industrial production.

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