September 29th 2016

Gaussia Luciferase / Toluene bioluminescence assay

Objectives

Establish a correlation between the concentration of toluene and the production of luciferase, thus of bioluminescence. We tested 4 different concentrations (10 ng/L, 100 ng/L, 10 µg/L and 10 ng/L) in 3 replicates in 4 different times of incubation with Toluene.

Materials

Toluene preparation:

Absolut liquid toluene : Sm = 867 g/L (244511 SIGMA-ALDRICH)
Dilution toluene :

S1 = 1 g/L, Vf1 = 20 mL —> Add 230 µL toluene Sm into 200 mL ethanol
S2 = 10 mg/L, Vf2 = 25 mL —> Add 2.9 µL toluene Sm into 250 mL ethanol
S3 = 10 µg/L, Vf3 = 25 mL —> Add 25 µL toluene S2 into 250 mL ethanol

BioLux Gaussia Luciferase Assay Kit (#3300S)

Protocol

Inoculation:

Resuspend 100 µL DH5⍺ bacteria O/N culture into 150 mL Liquid LB.
When OD is equal to 0.1, transfer the wanted volume of bacterial culture into 48 falcon tubes (4 different concentrations and 4-time measurement in 3 replicates)
Then add the wanted volume of toluene under the chemical hood starting by the less concentrated:

Controls:
- Negative control with LB only into 12 falcon tubes (4-time measurement in 3 replicates)
- Negative control without biosensor (LB+Toluene) into 12 falcon tubes (4-time measurement in 3 replicates)
- Negative control with BB12 : plasmid with XylR gene but without GLuc gene (Pr-RBS-XylR-Term)
  
  When OD = 0.1, transfer the wanted volume of bacterial culture into 12 falcon tubes (4-time measurement in 3 replicates)
- Positive control with G1 (Pr-RBS-GLuc-Term)
T0: Incubate the tubes (84 Falcon 50 mL tubes) of each cultures at 37°C and 250 rpm.

Bioluminescence measurement:

Measurement on 4 incubation durations:

T1: 11 am (1h)
T2: 1 pm (3h)
T3: 3 pm (4h30)
T4: 5 pm (5h)

We used the BioLux Gaussia Luciferase Assay Kit (#3300S).
The substrate is at 1X concentration recommended by the supplier NEB : BioLux Substrate #B3302A.

For each replicate of each incubation time :

Drop-off 20 µL of bacterial culture in each wells from the falcon tubes of one replicate (Controls + C1, C2, C3, C4 —> 8 wells).
Drop-off 50 µL of substrate solution in one well and start the bioluminescence measure of this well just after. Repeat the operation in each wells. In the program of measurement we add 3 seconds of shake before the luminescence measurement.