Team:Osaka/Protocols

Protocols

Cell survival assay 1: Mitomycin C

1. 10 ml of LB broth was inoculated with 100μl 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.

Cell survival assay 2: Hydrogen peroxide

1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.