Sterile breathable film

Growth inhibition was measured as previously described [15 (link),23 (link)]. Briefly, overnight cultures of MJF376, MJF451, MJF455, and MJF612 strains carrying constructs were diluted at 1:50 (v:v) in CphM and grown, until an OD₆₀₀ of 0.2 was reached. Expression was then induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 30 min, 10 mM stocks of compound 262 solubilized in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were diluted two times to achieve its final concentration in pre-warmed CphM with a final DMSO concentration of 2%, and 100 µL were added to wells of a pre-warmed, sterile 96-well flat bottom plate (Greiner bio-one, Monroe, NC, USA). Cultures were then diluted at 1:200 (v:v) in pre-warmed CphM, 100 µg/mL ampicillin, and 2 mM IPTG with diluted experimental antibiotics and were indicated, at 2× their concentration or mock (DMSO only). One hundred microliters of culture mixture with kanamycin A at a final concentrations of 0.2 µM (Sigma-Aldrich, St. Louis, MO, USA) were added to 96-well plates for a total of 200 µL, sealed with a sterile breathable film (Axygen, Union City, CA, USA), wrapped in aluminum foil and placed in a 37 °C shaker and rotated at 110 Cycles per minute for 16–17 h. OD₆₂₀ was then taken with a Multiskan Ascent 354 (Thermo Fisher Scientific, Waltham, MA, USA) plate reader.

Frozen stocks of the MJF455 strain carrying MscL single cysteine mutations (S2C-E107C) were made in a 96-well plate format (GOF and LOF mutations were omitted). This plate was used to inoculate overnight cultures grown in a flat bottomed 96-well plate covered with a sterile breathable film (Axygen) in CphM at 37°C at 170 rpm. OD₅₉₅ was taken the next morning to ensure all samples grew. 96-well plates containing 190 μl of prewarmed CphM, 1 mM IPTG or CphM, 1 mM IPTG, and DHS at 6.25 μm were inoculated with 10 ul from the overnight culture plate. Plates were sealed with breathable film, wrapped in foil and placed in a 37°C incubator without shaking for 16 h. OD₅₉₅ was then taken, and the blank was subtracted. The difference in OD₆₀₀ was calculated between the plate with DHS and without DHS. Hits were then selected by at least 4 times more growth compared to WT Ec-MscL.