Measuring B. subtilis Stress Response

A glycerol stock stored at –80°C containing B. subtilis with the desired genomic integration was streaked onto an LB plate supplemented with 5 μg/ml chloramphenicol and incubated overnight at 37°C. The next day, a single colony was inoculated in MGMM and incubated overnight at 37°C with agitation at 220 rpm. The following day, overnight cultures were diluted 1:10 into 1 ml fresh MGMM supplemented with the desired concentration of 10 μl 100 mM caffeine or 10 μl H₂O (mock treatment). Three technical replicates were prepared for each experimental condition. The cultures were incubated at 37°C with agitation at 220 rpm for 5 h. 100 μl aliquots were pipetted into a 96-well plate and OD₆₀₀ was measured. 100 μl uninoculated media was used as a blank. 50 μl of permeabilization buffer (100 mM Tris [pH 7.8 at ∼20°C], 32 mM Na₂HPO₄, 8 mM DTT, 8 mM cyclohexanediaminetetraacetic acid, 4% Triton X-100) supplemented with 0.75 mg/ml lysozyme was added to each well. After waiting 15 min, 50 μl of 4 mg/ml ortho-nitrophenyl-β-galactoside (ONPG) was added to each well. A plate reader was used to measure OD₄₂₀ at 1-min intervals over a 2 h period while incubating at 28°C. The blank OD₄₂₀ reading (from uninoculated media) was subtracted from the OD₄₂₀ reading of each sample at every timepoint. Specific β-galactosidase activity was calculated by determining the slope (OD₄₂₀/min) of the linear portion of the OD₄₂₀ versus time curve for each sample and dividing this value by the corresponding OD₆₀₀ reading. Statistical analysis was performed with a t-test (two-tailed distribution, two sample equal variance).