Cfrench:BacTrans1

Transformation of Bacillus subtilis

This protocol was developed by Lucy Montgomery based on published protocols (Anagnostopoulos and SPizizen, 1961, J. Bacteriol. 81:741 and Young and Spizizen, 1961, J. Bacteriol. 81:823). It has been used successfully with pVK168 (specR) and pTG262 (cmlR).

Materials needed

• Plate of B. subtilis 168
• 20% glucose
• 1.0 M MgSO4.7H2O
• 0.1 M CaCl2
• 0.05 M MnSO4
• 20% w/v casamino acids
• 11 mg/ml indole (dissolve in dimethyl sulfoxide)

5 x Spizizen's Salts

• available from the media prep room; if you need to make this for yourself, it contains:
• 1% w/v ammonium sulphate
• 7% w/v K2HPO4
• 3% w/v KH2PO4
• 0.5% w/v Na3citrate dihydrate
• 0.1% w/v magnesium sulphate)

Solution P

• 25 ml 1 M MgSO4
• 5 ml 0.1 M CaCl2
• 0.1 ml 0.05 M MnSO4
• 70 ml water

Pre-Transformation Medium (PTM)

• 2 ml 5 x Spizizen's Salts
• 8 ml water (sterile)
• 0.5 ml 20% glucose
• 0.1 ml solution P
• 0.2 ml 20% casamino acids
• 0.01 ml indole (11 mg/ml)(B. subtilis 168 is auxotrophic for tryptophan and requires indole or tryptophan to be provided).

Transformation Medium (TM)

• 2 ml 5 x Spizizen's Salts
• 8 ml water (sterile)
• 0.3 ml 20% glucose
• 5 microlitres 20% Casamino Acids
• 10 microlitres indole (11 mg/ml)

Procedure

• Grow an overnight culture of B. subtilis 168 in L-broth.
• Check the OD at 600 nm. The one time I tried this, it was 1.13, which seems lower than I expected.
• Prepare 10 ml PTM and place 5 ml in each of 2 vials (we use 40 ml glass universals).
• Inoculate each vial with overnight culture, one more heavily than the other. I used 0.5 ml and 1.0 ml. Lucy's protocol calls for final OD about 0.25 and 0.75 but mine were obviously considerably lower.
• Grow the cultures at 37 C with shaking until they get reasonably turbid. Lucy's protocol calls for an OD of 3, but my cultures did not reach this. After 5 hours they were at 2.12 and 1.79, at which point I got bored and used them anyway.
• As an alternative to monitoring OD, check motility by wet mount. I am informed that when B. subtilis is motile, it is competent. I checked my cultures at the time/OD noted above and both were vigorously motile.
• Prepare PTM and dispense 1 ml to each of however many vials you want to transform, not neglecting a negative control with no DNA.
• Add 0.1 ml culture to each vial, and 5 microlitres (about 0.5 micrograms or so) of plasmid DNA.
• Incubate with shaking at 37˚C for 30 to 90 minutes.
• Plate onto selective plates. Spectinomycin at 100 mg/l is used for pVK168, chloramphenicol at 10 mg/l for pTG262 and derivatives thereof.
• For pTG262 I obtained numerous chloramphenicol-resistant colonies after one night incubation. Constructs based on pVK168 and plated on spec100 seem to require longer to grow.