A single step three-strain in vivo Gateway reaction

We developed a simplified, highly efficient Gateway reaction that recombines target DNA to expression (destination) plasmids in vivo and subsequently conjugates the final vector into a recipient strain, all in a single step. This recipient strain does not need to contain any selective marker and can be freely chosen as long as it is sensitive to ccdB counterselection and can be targeted by the RP4α conjugation system. Our protocol is simple, robust, and cost effective. It works in 96-well plate format and performs across a range of temperatures. We designed modular, minimal destination vectors containing a modified Gateway insert to ease vector design by providing locations for insertion of tags, promoters, or conjugations. To demonstrate the utility of our system, we created destination vectors with split adenylate cyclase tags for bacterial two-hybrid (B2H) studies and screened a library of diguanylate cyclases for protein-protein interactions in a single step.