96 well black clear bottomed plate

Phage-based luciferase assays were performed as described previously.⁶³ (link) For each replicate, one colony of the evolution strain was grown overnight to saturation in DRM and appropriate antibiotics and then back-diluted 50-fold into DRM with appropriate antibiotics. Cultures were grown at 37°C with shaking at 230 RPM until cultures reached OD₆₀₀ = 0.4. The mid-log culture was distributed into a 96-well black clear-bottomed plate (Corning), 135 μL of culture per well. 15 μL of high-titer (1 x10¹¹ pfu/mL) phage were added to each well. The plate was covered with a breathable seal and incubated, shaking at 37°C and 230 RPM for 3.5 h. Luminescence and OD₆₀₀ were measured using a plate reader (TECAN). Values reported are OD₆₀₀-normalized luminescence.

Strains for plasmid-based luciferase assays were made by transforming chemicompetent S2060 E. coli with all necessary plasmids, recovering in antibiotic-free DRM for 2 h, and then plating on 2x YT agar containing maintenance antibiotics and 100 mM glucose. For each biological replicate, one colony was picked into DRM and grown overnight. The following day, cultures were back-diluted 50-fold into DRM and antibiotics. For induced samples, arabinose was added to a final concentration of 20 mM. Cultures were grown shaking at 230 RPM and 37°C for 3 h, after which 150 μL were removed, placed into a 96-well black clear-bottomed plate (Corning), and measured for luminescence and OD₆₀₀ on a plate reader (TECAN). Values reported are OD₆₀₀-normalized luminescence.

Recombinant strains based on NRRL B-3805 expressing the eGFP constructs were growing for 60 h in LB medium with kanamycin. Then, the cells from 1 mL of the bacterial culture were harvested by centrifugation at 2000 g for 1 min, washed with 1 mL of 10 mM K₂HPO₄ buffer (pH = 7.4) three times, and resuspended in the same buffer. Then, 200 µL aliquots were transferred into a clear-bottomed black 96-well plate (Costar) and the plate was placed in a multifunctional microplate reader (BioTek Synergy H1). Fluorescence was measured at an excitation wavelength of 488 nm and emission wavelength of 528 nm. The fluorescence values were normalized to the respective optical density at 600 nm (OD₆₀₀) of the cell suspension in each well.