METHOD FOR INTRODUCING UNMARKED POINT SUBSTITUTIONS INTO THE CHROMOSOME OF MICROBACTERIA Russian patent published in 2022 - IPC C12N15/00 C12N15/10 C12N15/11 

FIELD: biotechnology.

SUBSTANCE: invention relates to genetic engineering. Described is a method for introducing point substitutions into the bacterial chromosome without concomitant integration of the antibiotic resistance determinants. The method is based on the widely applied method of phage recombination for introducing mutations into the bacterial chromosome using short fragments. The efficiency of obtaining recombinant clones in Mycobacteria is approximately 10^-4 to 10^-3 per total amount of cells. Accordingly, selection of the appropriate mutants requires checking thousands of colonies. The previously proposed methods for improving the efficiency of clone selection either include the use of antibiotic resistance markers or include the creation of complex genetic constructs individually for each introduced replacement. The increase in the relative yield of recombinant colonies is achieved due to the transformation of the recombinant oligonucleotide jointly with the co-selective DNA incapable of being intagreated into the chromosome. The selection of colonies based on the phenotypic resistance provided by the resistance marker of the coselective DNA provides a possibility of selecting a competent subpopulation of cells and increasing the effectiveness of the method to 10^-2 to 10^-1 recombinants. The loss of an extra-chromosomal resistance marker is provided either by serial passages of the culture onto a medium without an antibiotic or by the use of counter-selective markers.

EFFECT: introduction of point substitutions into the bacterial chromosome.

9 cl, 2 dwg, 5 ex