B. subtilis growth curves

B. subtilis expressing genes of interest on the pHT01 plasmid (Nguyen et al., 2007) were struck on LB-chloramphenicol plates on day one and grown overnight at 30°C. On day 2, colonies were re-struck on LB-chloramphenicol with 1 mM IPTG agar plates overnight at 30°C. On day 3, biomass was scraped and processed as described in the ‘bacterial culturing’ section above. The bacteria were then normalized to an OD₆₀₀ of 1 and inoculated 1:100 into a 96-well plate containing TSB chloramphenicol and 0.5 mM IPTG. 4-HNE was then added to the bacteria at various concentrations and the bacteria were allowed to grow at 37°C in Synergy HTX plate reader for 12 hr with shaking.