Live-cell imaging of bacterial DNA repair

Cells were imaged in quartz-top flow cells as described previously^{23 (link)}. Briefly, flow cells were assembled using a clean quartz piece (Proscitech, Australia) and a bottom cleaned and (3-Aminopropyl)triethoxysilane (Alfa Aesar, A10668, UK)-treated coverslip (Marienfeld, Deckglaser, 24 mm × 50 mm, No. 1.5, German) using double-sided sticky tape (970XL ½ × 36 yd, 3 M, United States), and sealed with 5-min epoxy (Parfix). Quartz top pieces were designed to be able to insert inlet and outlet tubing (PE-60, Instech Labs). Prior to imaging, cells were revived from a −80 °C DMSO stock in 500 μL of EZ-rich defined media (Teknova, CA, US), supplemented with 0.2% (v/v) glucose in 2-mL microcentrifuge tubes at 30 °C. Cultures were set to shake in an Eppendorf Thermomixer C (Eppendorf, Australia) at 1000 rpm. On the following day, cultures were reset by inoculating fresh growth medium 1:200 fold, and continued to shake for ~3 h at 30 °C prior to imaging. For experiments involving plasmid-expressed UvrA-YPet or UvrA(Δ131–250)-YPet, spectinomycin (50 μg per mL) was added to the growth media. Cells in early exponential phase were loaded in flow cells at 30 °C, followed by a constant supply of aerated EZ-rich defined media at a rate of 30 µL per min, using a syringe pump (Adelab Scientific, Australia).