Method for establishing saccharomyces integrated gene mutation library based on in vivo homologous recombination

Abstract

A method for establishing a saccharomyces integrated gene mutation library based on in vivo homologous recombination belongs to the fields of protein engineering, molecular biology technology and genetic engineering. The method comprises the steps of establishing the recombination expression plasmid of the target gene, designing long primer fragment by taking the recombination expression plasmid as a template, amplifying error-prone PCR to obtain the mutated gene with the expression carrier 20-70bp homologous sequence at two ends, respectively carrying out the enzyme tangential linearization to the homologous region of two ends of the mutated gene of the expression carrier and the homologous region of the gene group, mixing the error-prone PCR amplified outcome and the linearized expression carrier by a definite molar ratio and carrying out electro-transformation of the saccharomyces, and integrating the exogenous mutation target gene into the saccharomyces gene group, thus obtaining gene mutation library. The method has high efficiency and convenient operation, shortens the library establishment period from 1-2 weeks to 3 days, needs any sub-cloning steps for library establishment, reduces the loss of content and richness of the gene mutation, has generalization and can be used for various target genes capable of being expressed in the saccharomyces.

Description

technical field [0001] The invention relates to a method for constructing a yeast integrated gene mutation library based on homologous recombination in vivo, and relates to a method for constructing a gene mutation library by homologous recombination. It belongs to the fields of protein engineering, molecular biology technology and genetic engineering. Background technique [0002] The directed evolution of enzymes in vitro belongs to the irrational design of proteins. It does not need to know the spatial structure and catalytic mechanism of enzymes in advance. By artificially creating special conditions and simulating the natural evolution mechanism (random mutation, recombination and natural selection), it can be transformed in vitro Enzyme genes, and directed selection of mutant enzymes with desired properties. The in vitro directed evolution technology of enzymes has greatly expanded the research and application scope of protein engineering, especially it can solve the ...

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Problems solved by technology

This process is cumbersome, inefficient, time-consuming and labor-intensive, and greatly reduces the capacity of the mutant gene. The gene mutation library needs to be screened from thousands of transformants to select the target transformant, so the traditional method cannot satisfy the construction The need for yeast integrative gene mutation libraries

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