Dandekar & Chandler:Screen for Bactobolin Resistant Mutants
Screen for Bactobolin Resistant Mutants
1. Streak WT B. subtilis onto LB and incubate at 37C.
- a. JH642: Transformable strain
- b. 3610: Wild strain that was able to mutate L2
2. Streak WT B. thailandensis onto LB and incubate at 37C.
1. Inoculate one colony into a tube with 5 mL LB.
2. Inoculate ~5 colonies of B. thailandensis into each of 3 baffled flasks with 10 mL LB.
3. Incubate all o/n at 30C shaking.
1. Harvest B. thailandensis supernatant:
- a. Pellet cells in microcentrifuge and keep supernatant
- b. Filter/sterilize supernatant twice with 0.22 uM filters
- c. Store supernatant at 4C
2. Pour LB plates supplemented with various amounts of supernatant. Aim for a concentration where growth is just inhibited.
- a. 100-150 uL supernatant per 20 mL LB agar worked best for this screen.
3. Spread 100 uL undiluted B. subtilis culture onto each plate.
4. Incubate at 30C for 1-2 days.
Day 4 or 5:
1. Restreak potential resistant isolates onto fresh supernatant plate alongside WT as a negative control. Incubate at 30C for 1-2 days.