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.