A method for identifying microbial gene functions based on inducible promoters

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a method for identifying microbial gene functions based on inducible promoters. The intergenic sequence upstream of the gene is selected, and the promoter prediction software is used to predict possible promoters, and the promoters to be used are obtained by comparison. subsequence; specifically design 4 specific primers for the promoter of the target gene to amplify the specific primers for the upstream and downstream homology arms; obtain the upstream and downstream homology arms; assemble the functional gene cassette; design primers to obtain Escherichia coli origin of replication; construction of homologous recombination plasmid; plasmid extraction; electrotransformation; positive clone verification; gene functional testing. The present invention is convenient and fast, and can be completed in batches in a relatively short period of time, including the microbial strain transformation of the present invention taking Zymomonas mobilis as an example, and completing gene identification and analysis horizontally and vertically; this experimental method can also be extended to other methods that can be passed through Homologous recombination realizes the bacterial transformation of gene fragment 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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