FIG. 4.

Site-, and orientation-specific integration of experimental plasmids mediated by ccrA and ccrB. PCR detection of the left and right plasmid-chromosome junctions was performed with four crisscross combinations of primers with DNAs extracted from the culture of N315ex(pSRatt), N315ex(pSRtta), and N315ex(pYT3att) as templates. Four primers L (cL1; see Fig. 1A), R (cR2; see Fig. 1A), α, and β were used. The location and direction of the four primers are illustrated. Only the regions adjacent to the introduced attSCC on the plasmids and the attB on N315ex chromosome are illustrated. The expected sizes of the amplified DNA fragments generated by a site-specific integration of the plasmids were 610 bp (L-α), 1,255 bp (L-β), 1,649 bp (R-α), 1004 bp (R-β), and 549 bp (L-R). The results show a strict site and orientation specificity of ccr-mediated integration. The molecular weight marker used was a 1-kb ladder.