Fig. 1. (Adapted from Blomfield et al. 1991).

The transfer of the sacB-kan cassette into the chromosome requires two recombination events leading to plasmid integration followed by plasmid excision. Step 1: plasmid integrates into the wild-type strain at the non permissive temperature for plasmid replication (42°C). Steps 2 and 3: plasmid integrates are grown at 28°C in the presence of kanamycin to enrich for bacteria that have excised and cured the plasmid, leaving the cassette on the chromosome. Step 4: Growth on media containing LBC or LBK. Bacteria that can only grow on LBK are successful constructs of the intermediate strain.