Method for identifying microbial gene functions on basis of inducible promoters

Abstract

The invention belongs to the technical field of gene engineering, and discloses a method for identifying microbial gene functions on basis of inducible promoters. The method for identifying microbialgene functions on basis of the inducible promoters comprises the following steps: selecting intergenic sequences from upstream genes, predicting possible promoters by using a promoter prediction software, and carrying out comparison so as to obtain promoter sequences for standby application; designing 4 specific primers according to promoter specificity of a target gene so as to be used for amplifying specific primers of upstream-and-downstream-homologous arms; obtaining upstream-and-downstream-homologous arms; assembling a functional gene cassette; design a primer so as to obtain a starting point of E.coli replication; constructing homologous recombinant plasmids; performing extraction on the plasmids; carrying out electrical conversion; carrying out positive clone verification; and carrying out gen function detection. The method for identifying microbial gene functions on basis of the inducible promoters is convenient, quick and capable of completing batch identification, including microbial strain transformation taking zymomonas mobilis as an example as well as transverse and longitudinal completion of gene identification and analysis, in a relatively short period of time; moreover, the experimental method can also be extended to other bacterial transformation work which can achieve gene fragment recombination by homologous recombination.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for identifying microbial gene functions based on an inducible promoter. Background technique [0002] At present, most of the existing technologies commonly used in the industry are to obtain the deletion mutant strain of the gene to be tested through homologous recombination, and then perform complementation experiments to verify the function of the gene. At the same time, the function of the gene is verified by overexpressing the target gene on the genome and plasmid. With the rapid development of large-scale sequencing and mass spectrometry technology, systems biology research methods are widely used in various fields of life science research, promoting genome-wide research and elucidating the correlation between genotype and phenotype. However, systems biology research usually brings a large number of target genes, which requires later high-throughput g...

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Problems solved by technology

[0007] (3) At the same time, although different overlap PCR methods have been developed and applied, the required synthetic primers have longer homologous ends and poor universality;

[0008] (4) Gene knockout and overexpression are mostly based on a single level, and the two analyzes cannot be performed simultaneously in situ

[0012] Overlap PCR experiment involves large variables, and no experiment proves its universality

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