Luciferase Assay Protocol for Bacterial Analysis

Luciferase assays were performed using a previously described methodology (22 (link), 29 (link)). Briefly, strains were diluted 1:40 from overnight liquid cultures and then incubated for 4 h at 37°C. Optical densities (OD₆₀₀) were subsequently measured for each sample to normalize luciferase values. Coelenterazine-H solution (Prolume) was added to each sample at a final concentration of 7.5 μg mL⁻¹, immediately followed by measuring the resulting luciferase activity using a Promega Glomax Discover luminometer. Normalized luciferase activity was determined by dividing luciferase relative light units (RLU) by the measured OD₆₀₀ values.