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.

Addendum 1 Sep 08

I have been playing around with this procedure and have made some aterations that seem to give higher transformation rates.

• Grow the initial overnight LB culture in a 100 ml flat bottle containing 5 ml LB. This provides better aeration than a 40 ml universal.
• Likewise, use 100 ml flat bottles for the PTM incubation step. I have also reduced the amount of indole added to 1 microlitre with no obvious harmful effects.
• Again, to improve aeration, do the actual transformation step in a 20 ml bijou bottle rather than a microcentrifuge tube. This gives both better aeration and better mixing.
• After plating out 100 microlitres on a chloramphenicol plate, you can transfer the remaining 1 ml or so of the transformation mixture to 5 ml of LB in a 100 ml flat bottle and add chloramphenicol to 5 micrograms/ml (ie add 1.25 microlitres of 20 mg/ml chloramphenicol stock solution). I have found that this concentration of chloramphenicol allows some growth of untransformed B. subtilis but growth of transformed cells is much faster, so incubating this overnight (at 37 C with shaking, like all other incubations in this procedure) allows transformed cells to grow out. You can then streak the solution to a chloramphenicol-10 plate the following morning if your main transformed plate shows no colonies.

Using this method, I have successfully transformed B. subtilis directly with a ligation of a BioBrick with pTG262 and obtained a large number of transformants (40 colonies on the 100 microlitre plate). When a few of these were tested by PCR, they showed the presence of the transgene as expected. This eliminates the requirement to initially transform E. coli with the ligation, then prep DNA and use this to transform B. subtilis, an annoying procedure since in my hands, pTG262 transforms lab strains of E. coli rather poorly.

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