M1000 instrument

For luminescence assays, cells were washed with DPBS, lysed with Reporter Lysis Buffer (Promega, Madison,WI), and 10 μL of whole cell lysate was added to 80 μL of luciferase assay buffer (Promega, Madison,WI) containing luciferin within a well of a 96 well optical bottom plate (Corning, Corning, NY). Plates were immediately loaded into a Tecan M1000 instrument and luciferase signal from each well was quantified. For fluorescence assays, cells were washed with DPBS and dislodged from the culture vessel by vigorous pipetting. Cells were resuspended in 50 μL of DPBS and added to individual wells of a 96 well optical bottom plate (Corning, Corning, NY) and YFP fluorescence (excite: 510 nm emit: 535 nm) for all wells was determined using an EnVision® Xcite Multilabel Reader (PerkinElmer).

For genome-wide competitive growth experiments, a single aliquot of the Dub-seq library in E. coli DH10B was thawed, inoculated into 25 ml of LB medium supplemented with chloramphenicol (30 µg/ml), and grown to mid-log phase. At mid-log phase, we collected cell pellets as a common reference for BarSeq (termed start or time-zero samples) and we used the remaining cells to set up competitive fitness assays under different experimental conditions at a starting OD600 of 0.02. For carbon source growth experiments, we used M9 defined medium supplemented with 0.3 mM l-leucine (as DH10B is auxotrophic for l-leucine)^{48 (link)} and chloramphenicol. For experiments with stress compounds, we used an inhibitory but sublethal concentration of each compound, as determined previously^{15 (link)}. All stress experiments were done in LB with chloramphenicol. All pooled fitness experiments were performed in 24-well microplates with 1.2 ml of media per well and grown in a multitron shaker. We took OD readings periodically in a Tecan M1000 instrument to ensure that the cells were growing and to confirm growth inhibition for the stress experiments. The assayed Dub-seq library cell pellets were stored at –80 °C prior to plasmid DNA extraction.