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- One culture, inoculated from a single colony was grown for 15hrs in M9salts(Bio101Inc.) with 0.005%(w/v) Casamino acids, 0.1%(v/v)glycerol, 2nMMgSO4, 0.1mM CaCl2 and kanamycin(20μg/ml) at 370C with shaking at 70rpm.
- Culture was diluted into fresh medium and allowed to grow for an additional 5 hours under the same conditions.
- 200μ l of the culture was transferred into flat-bottom 96 well plates (Greiner). Wells were pre-filled with the inducer molecule AHL at 8 final concentrations (0nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1μ M, 10μ M). .
- These plates were used to grow the cultures in a Wallac Victor3 multi-well fluorimeter at 370 C and assayed with an automatically repeating protocol of absorbance measurements (600nm filter, 0.1 second absorbance through approximately 0.5cm of fluid), fluorescence readings (488nm excitation filter, 525nm emission filter, 0.5 second, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed). Time between measurements was 3 minutes.
- Repeats were grouped into groups of three (four groups of three) and average of each group was calculated. Coefficient of variation between the four groups were calculated for different AHL concentrations.