Formylation gene cassette knockout mutant of Escherichia coli DH5alpha and construction method thereof

Abstract

The invention discloses a formylation gene cassette knockout mutant of Escherichia coli DH5alpha and a construction method thereof. The construction method utilizes a lambdaRed homologous recombination technology to simultaneously perform gene knockout on the gene fmt encoding the methionine-tRNA transformylase on the Escherichia coli E.coli DH5alpha chromosome and the gene def encoding the peptide deformylase, and PCR product electrophoresis and sequencing identification confirm that the obtained strain is a knockout mutant. The construction method of the invention is simple, an expression frame of other proteins on the genome is not changed, and the knock-in replacement of the chloramphenicol resistance gene facilitates the preservation of the strain and is beneficial for subsequent application. The constructed formylation gene knockout mutant of Escherichia coli DH5alpha completely eliminates the influence of PDF from the gene level, and can be used as the original material for studying the physiological process of formylation and deformylation, and the samples are provided for proteomics and metabolomics.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a formylation gene cassette (def-fmt) knockout mutant strain of Escherichia coli DH5α and a construction method thereof. Background technique [0002] Escherichia coli DH5α (abbreviated as E.coli DH5α) is an important model engineering strain and a derivative strain of Escherichia coli K-12, a commonly used recipient cell in molecular cloning technology. It plays an important role in research fields such as genetics. [0003] The homologous recombination technology is commonly used in the gene knockout process of engineering bacteria, and the frequency of homologous recombination is much lower than that of non-homologous recombination. At present, the commonly used method for knocking out the target gene is the Red recombination system relying on λ phage, which consists of three genes: gam, bet, and exo. The gam gene product is Gam protein with a molecul...

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Problems solved by technology

However, for this energy-consuming physiological process (formylation-deformylation), there is no direct evidence to prove its physiological significance

Bienvenut et al. (Proteomics 2015, 15, 2503–2518) performed proteomic analysis on the N-terminus of E. coli protein amino acids. However, they used a PDF inhibitor, which did not fundamentally eliminate the influence of PDF on the N-terminus of the protein, so The results obtained are by chance

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