Team:Osaka/Protocols

Protocols

Cell survival assay: Mitomycin C and Hydrogen peroxide

1. 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown for 2 h at 37°C.
2. Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h.
3. Cells were split into 3 ml aliquots in test tubes, and various concentrations of antibacterial agents were added.
4. After incubation for 2 h with shaking, centrifuge and discard antibacterial agents spiked medium and resuspend with fresh LB medium.
5. [RECOMMENDED] Dilute pre-culture samples according to expected survival rate, as determined from a preliminary experiment (lower dilution rate is needed if fewer cells are expected to survive).
6. Pipette 100 µl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
7. Wrap plates in aluminium foil and incubate at 37°C.
8. After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.

Promoter assay : Dual-GloR Luciferase Assay System (Promega)

1. 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown for 2 h at 37°C.
2. Cells were split into 3 ml aliquots in test tubes, and various concentrations of antibacterial agents were added.
3. After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake.
4. Decant supernatant, wash with 1mL PBS
5. Centrifuge at 3,000g for 5 min with soft brake.
6. Decant supernatant, add 100 µl cell lysis buffer.
7. Remove lysate 10-50 µl to the vial.
8. Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
9. Wait at least 5 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
10. Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
11. Wait at least 5 minutes, then measure Renilla luminescence
12. Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.