METHOD FOR INTRODUCING AN EXOGENOUS DNA BY OVERCOMING THE RESTRICTION MODIFICATION BARRIER OF A TARGET BACTERIUM

摘要

The present invention discloses a method for introducing an exogenous DNA by overcoming the restriction modification barrier of the target bacterium. The method provided in the present invention comprises the steps of 1) co-expressing all DNA-methyltransferase-encoding genes in the genome of the target bacterium in E. coli in which the restriction modification system thereof has been deleted to obtain a recombinant bacterium A; 2) introducing an exogenous DNA molecule into the recombinant bacterium A for in vivo modification so as to obtain a methylation-modified exogenous DNA molecule; 3) introducing the methylation-modified exogenous DNA molecule into the target bacterium. The experiments of the invention have demonstrated that the invention has a high transformation efficiency compared to prior methods for enabling genetic manipulation by overcoming the restriction modification barrier of the bacterium.

主权项

1. A method for introducing an exogenous DNA molecule into a target bacterium, comprising steps of:
1) co-expressing, in E. coli in which the restriction modification system thereof has been deleted, all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases in the genome of the target bacterium to obtain a recombinant bacterium A which shows the same DNA methylation pattern as that of the target bacterium; 2) introducing an exogenous DNA molecule into said recombinant bacterium A for in vivo modification to obtain a methylated exogenous DNA molecule; and 3) introducing said methylated exogenous DNA molecule into said target bacterium, wherein the E. coli in which the restriction modification system thereof has been deleted is derived from an original strain of E. coli deficient in recA gene by repairing the deficient recA gene and knocking out dcm gene and dam gene.