Luciferase activities of strains harbouring pBS3Clux-derivates were assayed using a Synergy™2 multi-mode microplate reader from BioTek® (Winooski, VT, USA). The reader was controlled using the software Gen5™. Culture volumes were 100 μl per well in 96-well plates (black walls, clear bottom; Greiner Bio-One, Frickenhausen, Germany), and incubation occurred at 37°C with agitation (intensity: medium). Cell growth was monitored by optical density at 600 nm wavelength (OD600). Raw luminescence output (relative luminescence units, RLU) was normalized to cell density by dividing each data-point by its corresponding corrected OD600 value (RLU/OD).
For constitutive promoters, LB or MCSE medium was inoculated 1:500 from overnight cultures of each strain. Cultures were incubated at 37°C with agitation and OD600 as well as luminescence were monitored every 10 min for at least 13 hours. For inducible promoters, 10 ml of LB or MCSE medium were inoculated 1:500 from overnight cultures and grown to OD600 = 0.2-0.5. Those pre-cultures were diluted to OD600 = 0.05 (MCSE) or 0.01 (LB), respectively, and transferred to eight (PliaI) or ten (PxylA) wells of a 96-well plate. The OD600 as well as luminescence were monitored every 10 min for one hour. At an OD600 ~ 0.1 (corresponding to OD600 ~ 0.4 in cuvettes of 1 cm light path length) 5 μl of the inducer was added to final concentrations of 0.1, 0.3, 1, 3, 10, 30 or 100 μg ml-1 Zn2+-bacitracin or 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.07, 0.2, 0.5% (w/v) xylose. To one culture-well, 5 μl of water was added as uninduced control. Cultures were incubated at 37°C with agitation and the OD600 as well as luminescence were monitored every 10 min for at least 13 hours.
For constitutive promoters, LB or MCSE medium was inoculated 1:500 from overnight cultures of each strain. Cultures were incubated at 37°C with agitation and OD600 as well as luminescence were monitored every 10 min for at least 13 hours. For inducible promoters, 10 ml of LB or MCSE medium were inoculated 1:500 from overnight cultures and grown to OD600 = 0.2-0.5. Those pre-cultures were diluted to OD600 = 0.05 (MCSE) or 0.01 (LB), respectively, and transferred to eight (PliaI) or ten (PxylA) wells of a 96-well plate. The OD600 as well as luminescence were monitored every 10 min for one hour. At an OD600 ~ 0.1 (corresponding to OD600 ~ 0.4 in cuvettes of 1 cm light path length) 5 μl of the inducer was added to final concentrations of 0.1, 0.3, 1, 3, 10, 30 or 100 μg ml-1 Zn2+-bacitracin or 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.07, 0.2, 0.5% (w/v) xylose. To one culture-well, 5 μl of water was added as uninduced control. Cultures were incubated at 37°C with agitation and the OD600 as well as luminescence were monitored every 10 min for at least 13 hours.
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