Figure 8

Overview of cell-based ASST sulfotransferase assay. (A) Bacterial strains were cultured in LB-Lennox media overnight at 37 °C with aeration (200 rpm). (B) Growth analysis plates were prepared by subculturing the O/N cultures from (A) into a 96-well plate containing the test inhibitors (akin to preparing an MIC challenge plate). (C) Growth plates were incubated at 37 °C, 300 rpm, in a microplate reader programmed to take O.D. 600 nm readings every 15 min for 15 h. (D) Growth plate cultures were transferred in a fresh 96-well plate with each culture well normalised at an O.D. 600 nm of 0.4. (E) A master mix containing 4 µL of phenol (50 mM), 10 µL of MUS (10 mM), and 126 µL of LB-Lennox per reaction well was prepared. (F) The reaction master mix (140 µL) was added to normalized cultures (60 µL) and mixed. (G) Fluorescence at 450–480 nm was immediately monitored in a plate reader (BMG, Australia) with measurements obtained every 5 min for up to 90 min. (H): Data acquisition and analysis.