Replication is required for Red recombination. (A) Schematic illustration of an experiment to replace the oriγ replication origin in an R6K-amp plasmid with the pBR322 replication origin and the bsd gene for blasticidin resistance. (B) Quantification of recombinant colonies from the experiment shown in (A). Recombination only occurred when the pir gene was expressed in trans from the E. coli chromosome (strain GB2005-pir). No recombination product was found in the absence of pir (strain GB2005). (C) Schematic illustration of an experiment to rebuild the pir gene in an R6K plasmid using an ampicillin resistance gene bla and a 5' fragment of the pir gene, which completes the pir gene when fused with the 3' end of the pir gene. (D) Quantification of recombinant colonies of the experiment depicted in (C). Similar to the result shown in (B), recombination only occurred when the Pi protein was expressed in trans.