Oligonucleotide mediated colibacillary gene knock-out or point mutation method

Abstract

The invention relates to a method for carrying out gene knock-out or point mutation on a colibacillary genome by an oligonucleotide mediated recombineering measure. The method comprises the following steps: firstly, integrating a gene box of a cane sugar 6-fructosyltransferase gene and a kanamycin resistance gene containing isogenous arms into a target gene through recombineering; and then, carrying out isogenous recombination on oligonucleotide containing the isogenous arms and the isogenous sequences on the genome through the recombineering to remove the cane sugar 6-fructosyltransferase gene and the kanamycin resistance gene. Thereby, the gene knock-out and point mutation without any basic group redundance are realized. The gene knock-out oligonucleotide design conforms to the basic group sequences of both sides of the target genes, and base groups needing the mutation are introduced in the oligonucleotide by the point mutation oligonucleotide. The invention does not need in-vitro clone and in-vitro realization of some basic group bit mutation or mutation gene transplanting into germ bodies. The method of the invention can provide an effective early-stage operation platform for the industrial production and the research such as genetics, molecular biology, biochemistry and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for gene knockout or point mutation of Escherichia coli mediated by oligonucleotides. Background technique [0002] Escherichia coli is an important tool for research in genetics, molecular biology, biochemistry, etc. Its typical features are: rapid growth (doubling time 17 minutes), clear genetic background, diverse genetic manipulation methods, and the genomes of various Escherichia coli have been analyzed, etc. Escherichia coli is also one of the most important strains for industrial production of various active proteins, metabolites (such as amino acids, pigments and alkaloids) and secondary metabolites (such as physiologically active substances). In view of the importance of Escherichia coli, it is very important to develop fast and easy genetic manipulation methods for modifying the genes of Escherichia coli, and gene knockout and point mutation are two key tech...

AI Technical Summary

Problems solved by technology

The existence of redundant sequences may interfere with the overall structure of the genome and affect the expression of other genes, such as polarity effects (and the expression of downstream genes in the same transcription direction are affected), which also brings difficulties to subsequent genetic modification

[0007] In order to achieve genetic modification of E. coli without any redundant bases, Yu et al. first cut the homologous fragment used for genetic modification, resistance gene, I-SceI enzyme and I-SceI enzyme that is not contained in the E. coli genome The site sequence is introduced into the E. coli genome, and the I-SceI enzyme induced by inactivated chlortetracycline catalyzes the homologous recombination of the homologous fragments on both sides of the I-SceI restriction site to include the resistance gene (Yu, B.J., et al., Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Res, 2008.36 (14): e84.) The advantage of this method is to realize the removal of redundant sequences and large fragment gene knockout, the disadvantage is that it still needs to construct 400-500bp homologous fragments for gene modification by PCR, restriction enzyme digestion, ligase-mediated DNA fragment connection and other steps, which increases the time and difficulty

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