Allegra x 12r benchtop centrifuge

After incubation period of 24 h, plates were centrifuged (Allegra™ X-12R Benchtop centrifuge, Beckman Coulter Inc., Fullerton, CA, USA) at 3000 rpm for 10 min to precipitate the violacein. Supernatants were discarded from the wells by pipetting, and pellets were re-suspended in 200 μL of 96% ethanol by scraping and mixing followed by centrifugation (3000 rpm, 10 min). Thereafter, 100 μL of supernatants were transferred to sterile 96-microtiter well plates, and violacein was quantified by reading the optical density at 595 nm using Varioskan Flash. Inhibition of violacein production was expressed as inhibition percentages of the untreated biofilms (Equation (1)): in which untreated control = C. violaceum + DMSO, media control = LBY, and sample = C. violaceum treated with a compound. Later on, violacein extraction assay was also utilized for the determination of half inhibitory concentrations of the compounds.

1 mL LB media (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl) containing 10 µg/mL tetracycline was added to deep 24-well plates, and inoculated with single colonies from agar plates and grown at 37 °C and 750 rpm. 100 µL pre-culture was used to inoculate 4 mL 2YT media (1.6% (w/v) tryptone, 1% (w/v) yeast extract and 0.5% (w/v) NaCl) with 10 µg/mL tetracycline. Cells were incubated for 3–4 h to reach log phase, prior to the induction of expression with 0.1% (v/v) d-xylose (Sigma-Aldrich, St. Louis, MO, USA) for 16–20 h at 20 °C (GFP) or 37 °C (proteases) and 750 rpm. Cells were harvested using an Allegra X-12R benchtop centrifuge (Beckman Coulter, Brea, CA, USA) at 4750 rpm for 15 min. Proteins in 1 mL supernatants were precipitated with trichloroacetic acid (TCA; 10% final concentration) for 1 h at 37 °C, washed twice in 500 µL acetone, and resuspended in 40 µL 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis [32 (link)]. Cells were resuspended in 1 mL 8.5 N lysis buffer (50 mM Tris HCl pH 8.5, 50 mM NaCl, 0.25 mg/mL lysozyme, 10% (v/v) glycerol), and lysed by ultrasound as previously described [16 (link)]. Cleared lysates (soluble fraction) and pellets (insoluble fraction) were analyzed by SDS-PAGE [32 (link)].