Breathe easy sealing membrane

Bioassays were performed in M9-suc for two reasons. (i) OryR accumulated in X. oryzae when grown in rich medium (peptone-yeast extract-salts) in the absence of plant macerates (24 (link)), suggesting that something in complex medium can induce the system. Therefore, we decided to use a minimal medium so as not to confound our results. (ii) Succinate was chosen as the carbon and energy source in the minimal medium because there were no significant growth rate differences between the wild-type and pipR mutant strains in this medium. Strains containing pP_pipA-gfp were incubated overnight (24 h) in M9-suc plus kanamycin at 30°C with shaking. Cells were diluted 1:100 into fresh medium, 150-µl aliquots were added to individual wells of a 96-well microtiter dish containing 7.5 µl (except as indicated in Fig. 3) of material to be tested (leaf macerates, peptone, peptides, or AHLs), and the plates were sealed with Breathe-Easy sealing membrane (Research Products International, Mount Prospect, IL) and incubated at room temperature for ~24 h. GFP fluorescence (excitation at 485 nm and emission at 535 nm) and growth (OD₅₉₅) were assessed using a Tecan Genios pro plate reader, and data were plotted as relative fluorescence units (RFU) per OD unit.

BL21 (DE3) star E. coli (Invitrogen, Eugene, OR) were transformed with the identified bacterial expression vectors and grown for 18 h at 37 °C in Luria-Bertani broth (LB) with 100 μg/mL ampicillin. The cultures were then diluted 100 times and grown at 37 °C to OD = 1.0. The cells were again diluted 100 times (or as identified for the experiment) in LB and grown for 1 h with 1 mM IPTG at 37 °C. Aminoglycosides were added according to the experimental protocol and the cells were grown for 12 h at 37 °C in a Biotek Synergy plate reader sealed with Breathe-Easy sealing membrane (Research Products International Corp). To obtain growth curves, the OD600 was taken every 5, 20 or 60 min, depending on the experiment.
Saccharomyces cerevisiae BY4735 were transformed with the identified yeast expression vectors as previously described^{17 (link)}. The transformed cells were grown in SD-Uracil media in presence of 2% of glucose or raffinose for 19 h at 30 °C. Cells were diluted to A⁶⁰⁰ = 0.2 and Geneticin (G418) was added and grown in SD-Uracil media with 2% glucose or 2% galactose, 1% raffinose in Biotek Synergy plate reader for 30 h at 30 °C. Absorption at 600 nm (A⁶⁰⁰) was determined every 1 h for up to 30 h.

For in vivo characterization of CAT_Sa and its variants in E. coli, high-cell density cultures were performed as previously described [53 (link)] with an addition of 2 g/L of various alcohols. For in situ extraction of esters, each tube was overlaid with 25% (v/v) hexadecane. To confirm the protein expression of CAT_Sa and its variants, 1% (v/v) of stock cells were grown overnight at 37 °C and 200 rpm in 15 mL culture tubes containing 5 mL of LB medium and antibiotic. Then, 4% (v/v) of the overnight cultures were transferred into 1 mL of LB medium containing antibiotic in a 24-well microplate. The cultures were grown at 37 °C and 350 rpm using an incubating microplate shaker (Fisher Scientific, PA, USA) until OD reached to 0.4–0.6 and then induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h with a Breathe-Easy Sealing Membrane to prevent evaporation and cross contamination (cat# 50-550-304, Research Products International Corp., IL, USA). The protein samples were obtained using the B-PER complete reagent (cat# 89822, Thermo Scientific, MA, USA), according to the manufacturer’s instruction and analyzed by SDS-PAGE.