Bio one 655001

Biological triplicates of overnight pre-cultures were diluted 1:100 in 200 μL MOPS medium (50 mM MOPS, 43 mM sodium chloride, 93 mM ammonium chloride, 2.2 mM monobasic potassium phosphate, 1 mM magnesium sulfate), containing carbon sources as indicated, in a flat bottom, black polystyrene 96-well plate (Greiner Bio-One 655001). Ferrous sulfate was added, at a concentration of 1 µg/mL (3.5 µM), to the MOPS medium unless otherwise indicated. Carbon sources (succinate, lactate, glycolate, etc.) were added as sodium salts at the concentration indicated. Plates were incubated at 37°C with continuous shaking on the medium setting in a Biotek Synergy H1 plate reader. Expression of mScarlet, GFP, or luciferase was assessed by taking fluorescence or luminescence readings every 30 min for up to 24 h. mScarlet was detected at excitation and emission wavelengths of 569 nm and 599 nm, respectively. GFP was detected at excitation and emission wavelengths of 480 nm and 510 nm, respectively. Growth was assessed by taking OD readings at 500 nm simultaneously with the fluorescence/luminescence readings. Unless otherwise stated, the reported values were obtained by first subtracting the fluorescence values of “no-reporter” strains and then normalizing to growth (OD at 500 nm).