FIGURE 6.

Rationale of the recombineering method of scarless mutagenesis. (A) chloramphenicol resistance cassette (cat) and an I-SceI recognition site were PCR amplified from plasmid pKD3 using primers with 40 bp homology extensions (red and green rectangles) and were integrated within a target gene “orfA” via Red-mediated recombination. The plasmid pWRG99 (containing the lambda Red recombinase and I-SceI endonuclease) and a 200–500 bp dsDNA homologous recombination fragment were transformed by electroporation sequentially. (A) Site-directed mutagenesis of the region adjacent to the I-SceI recognition site and the resistance cassette. With the help of Red-mediated recombination, homologous recombination occurred between the 200 and 500bp dsDNA fragment harboring the same homologous regions (red and green rectangles) and the corresponding sequence of K88. (B) The bacteria would be killed if homologous recombination did not occur. The I-SceI endonuclease expressed from plasmid pWRG99 would kill unsuccessful recombinants by induction of double-strand breaks.