Dandekar & Chandler:Screen for Bactobolin Resistant Mutants

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Screen for Bactobolin Resistant Mutants

Day 1:

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.

Day 2:

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.

Day 3:

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.