Fig. 4
Cointegrate formation promoted by SGI1 Res. (a) Schematic maps of the res-site-containing parental plasmids (P1 = pJKI1088 and P2 = pJKI1092) and the expected structure of their cointegrate (C = pJKI1099) formed via site-specific recombination between res sites. Replication origins are indicated by white (p15A) and grey (R6Kγ) rectangles, resistance markers by blue (Km) and red (Cm) arrows, and the restored res sites by purple arrows. Direction of oligonucleotide primers used for amplification of junctions in the cointegrate are shown by small arrows marked with lower case: a – FRTfor, b – cat3.2, c – pucrev25, d – pBRBgl. (b) A representative set of colony PCR results from KmRCmRApS TG1 transformants obtained from transformation of plasmid DNA isolated from TG2 λpir and S17-1 λpir hosts, enabling replication of both parental plasmids and the resolvase-producing (Res+, pAVE20) or non-producing (Res−, pKK223-3) control plasmids. The TG1 host does not support replication of R6Kγ-based parental plasmid P2, thus TG1 transformants selected for the resistance markers of both parental plasmids (KmRCmR) should be cointegrates. Resolvase-producing (Res+) or non-producing (Res−) TG2 λpir and S17-1 λpir indicate the origin of the plasmid DNA introduced into the TG1 host. The junction regions of cointegrates were amplified using primer pairs a-d and b-c. The expected sizes of amplicons specific for the correct junctions are indicated. (c) Restriction mapping of cointegrates isolated from KmRCmR TG1 transformants that showed different patterns of amplicons in colony PCR screening. Correct cointegrate structures are marked by asterisks. Mw – molecular-weight marker (λ DNA digested with PstI) used in all gels. Original gels are presented in (Supplementary Fig. S8).
