Warning: in order to get proper result analysis, it is better to perform 3 replicates for each toluene concentration

Toluene preparation: stock solutions

100mg/L: Dilution of absolute liquid toluene to obtain a final toluene concentration at 100mg/L: Add 11,53uL of toluene into 100mL water (calculations done from toluene's molecular weight)
100µg/L: Dilute 10µL of the first stock solution into 10mL of water

Inoculation

From 1 UFC: Resuspend colony in 20 mL of LB medium containing chloramphenicol at 25µg/mL but free of toluene and grow over night.
When turbidity is high, inoculate the appropriate volume of bacteria culture to liquid LB medium containing chloramphenicol to reach 0,1 OD in the flask (100mL). Incubate culture at 37°C and 250 rpm .
When the OD reach 0.3, separate the initial bacterial culture to several falcon tubes (5mL in each tube) and add toluene to reach the desired toluene concentration.

Incubate culture at 37°C and 250 rpm.
Prepare the GLuc assay solution (e.g. 100 samples) by adding 50 μl of BioLux GLuc Substrate to 5 ml of BioLux GLuc Assay Buffer immediately before performing the assay. Mix well by inverting the tube several times (Do not vortex). Set the camera.
At several time (t= 30min, t=1h, t=1h30…, t=4h), pipet samples (5–20 μl per well) into a 96-well white (opaque) or black plate, or a luminometer tube.
Add the GLuc assay solution (50 μl) to a sample (i.e. Add the assay solution to only one sample at a time) and promptly measure the luminescence.
NB : Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate.