FIG. 3.

Construction of the LM-49 revertant via homologous recombination. (A) Flow chart of LM-49 revertant construction (1). In the LM-49 strain harboring the gene replacement plasmid pKSV7-AB, the plasmid may integrate into the host chromosome at either site a or site b (Pop in) (2). Four possible outcomes of pop-out recombination are shown. The PCR primer sets P1+P2, M13fwd+C1, and M13rev+C2 were used for distinguishing between these recombinations by PCR. (B) Identification of pKSV7-AB integration by colony PCR. A total of 30 colonies were purified and tested. The results from eight of them are shown. M, 1-kb DNA ladder marker; lanes 1 to 8, purified transformants PCR checked with M13fwd+C1 or M13rev+C2 as primers. (C) Analysis of plasmid and Tn917 junctions for checking type I and II colonies by colony PCR. (D) PCR identification of the revertant LM-49RE.