Selection stringency analysis
Selection stringency measurement is according to the report of Khetrapal with some minor modifications (Khetrapal et al., 2015). In brief, overnight cultures were 1:50 diluted and grown at 30 °C to OD600=0.4~0.6. 104-105 diluted cultures were spread onto non-restrictive LB agar plates and grown at 30 °C to quantify the number of surviving bacterial (CFU) in the overnight cultures. At the same time, about 1010 re-suspended cells after centrifugation were spread onto appropriate restrictive plates or placed at specific counter-selection conditions to quantify the number of colonies which escape the lethal effect of counter-selection gene.
Selection stringency frequency was determined by dividing the number of escaping colonies on restrictive plates by the CFU that grew on non-restrictive conditions.
Plasmid curing and electroporation
For the curing of plasmid pSim6, cells carrying pSim6 was inoculated into 2 ml of LB without ampicillin addition and incubated for 12 to 16 h at 37 °C. Cells equivalent in quantity to 10−4 μL of cultures were spread onto LB plates and grown at 37 °C. The colonies were confirmed as cured by determining their sensitivity to ampicillin (1 mg/mL for S. marcescens and 100 μg/mL for E. coli ) and by PCR identification of the elimination of pSim6.
Plasmid pKDsg-ack was then electroporated into the above pSim6-eliminated cells and spread onto LB agar plates supplemented with ampicillin (1 mg/mL for S. marcescens and 100 μg/mL for E. coli ).