Chemically competent cell preparation

Chemically competent cells were used for all cloning and assay pipelines. A glycerol stock of the appropriate strain was used to start a 2 mL culture supplemented with the appropriate antibiotics and grown up overnight at 30 °C at 300 RPM. The saturated culture was diluted 1:1000 in 50 mL 2xYT (United States Biological) with appropriate antibiotics and grown to OD₆₀₀ = 0.3–0.5 in a 37 °C shaker at 300 RPM. Cells were pelleted in a pre-chilled conical tube (VWR) by centrifugation at 8000 g for 10 min at 4 °C. The supernatant was removed and the cells were resuspended in approximately 20 mL 10% glycerol, then pelleted by centrifugation at 8000 g for 10 min at 4 °C. The supernatant was removed and the cells were resuspended in chilled TSS buffer (2xYT media supplemented with 5% DMSO, 10% PEG2250, 2 mM MgCl2). Cells were flash frozen in liquid N₂ at 100 µL aliquots and transferred to −80 °C storage.