Site-specific integration assay.

(A) Scheme of the plasmids used in the assay and the cointegrate molecule obtained. The donor suicide plasmid pCMS11 (pR6K::oriTp oriTw) is represented with a dotted line (left panel). oriTp and oriTw, the origins of transfer recognized by the relaxases of plasmids RP4 and R388 respectively, are shown as boxes in grey and black, respectively. nic sites are indicated with triangles in the respective colour code. The chloramphenicol resistance gene is represented as a white arrow (CmR). Recipient plasmid (pLA32) containing oriTw is shown as a black line (center panel). The last plasmid is the result of the integration reaction and is referred to as the cointegrate molecule (right panel). Relevant restriction sites are indicated in each plasmid. P1 and P2 are the oligonucleotides used to amplify the DNA of the integrants. (B) Representation of the integration assay in which the integrase is expressed in the recipient bacteria. The suicide plasmid is mobilized by the TraI relaxase (grey diamond) from the RP4 conjugative system (represented by a square) in a S17.1λpir donor strain to a DH5α recipient harbouring the oriTw-containing plasmid. Four different assays are represented: (i) no expression of integrases in the recipient bacteria (negative control); (ii-iv) recipient bacteria contain a plasmid coding for the relaxase domain of TrwC (N293-TrwC), full-length TrwC, or the TrwC/Rep chimera, respectively. TrwC protein is represented as black ellipses, and the Rep domain, in light gray. Integration frequencies obtained are also shown in the figure. Data are the mean of five independent experiments. Frequency is given as integrants/donors. (C) PCR analysis using primers P1 and P2. Agarose gel showing PCR amplicons (1.2 kb) obtained as a result of the integration reaction mediated by TrwC or TrwC/Rep chimera. RP, recipient plasmid; DP, donor plasmid; HP1, helper plasmid coding for TrwC protein; HP2, helper plasmid for TrwC/Rep chimera expression; M (1kb ladder): 10-8-6-5-4-3-2-1.5–1 kb.