Team:Osaka/Protocols

Protocols

Cell survival assay

1. 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD₆₀₀ reaches 0.4-0.6.
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 (we used Mitomycin C and Hydrogen peroxide) 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 until the OD₆₀₀ reaches 0.4-0.6.
2. Cells were split into 3 ml aliquots in test tubes, and various concentrations of antibacterial agents (we used Mitomycin C and Hydrogen peroxide) 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 10 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 10 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.