Fig. 2. (Adapted from Blomfield et al. 1991).

Step 1: Plasmid integration in the intermediate strain is selected at the non permissive temperature for plasmid replication (42°C). Steps 2 and 3: Plasmid integrates are grown at 28°C in the absence of antibiotics to enrich for bacteria that have excised (Step 2) and later cured (Step 3) the integrated plasmid. Step 4: Growth on media containing sucrose selects for successful strain constructs.