Assessment of the lethal effect
Streak cultivation was performed to analyze lethal effect of counter-selection marker under the control of pL promoter: colony grown well at 30 °C were streak-cultured onto
plate and placed at 42 °C to observe the lethal effect, and another plate with the same treatment was placed at 30 °C as a control.
To assess the lethal effect of counter-selection marker under the control of AraC/PBAD inducible system, colony was inoculated into 2 ml of LB broth supplemented with or without 0.4% arabinose and incubated for 12 to 16 h.
Spot titers experiment was performed according to previous report (Haeusser et al., 2014). In short, overnight culture was serially 10-fold diluted and 10 μL of diluted culture as indicated in the figures was spotted on the plates under corresponding counter-selection conditions, and another plate without the stress of counter-selection was used as a control.