Cfrench:BacTrans2

Generation of Bacillus subtilis competent cells (Bron, 1996)

N.B. This protocol assumes the use of B. subtilis 168 trpC2, which requires supplementation of minimal medium with tryptophan. For prototrophs this can be omitted, and for additional auxotropies the appropriate nutrients should be added.

1) Inoculate cells from a single fresh colony into 10 ml minimal medium (in a 250 ml conical flask).

2) Grow cells at 37°C with shaking at 200 rpm for 18 hours.

3) Subculture 1.4 ml into 10 ml prewarmed, fresh minimal medium and continue growth for 3 hours.

4) Add 11 ml starvation medium, continue growth for 2 h 45 min.

5) Either add sterile glycerol to 10% (v/v), dispense 0.3 ml aliquots and snap freeze in liquid nitrogen or continue directly with transformation.

Transformation of B. subtilis competent cells

For chromosomal integration of plasmid DNA by double-crossover, linearise plasmid by restriction endonuclease digestion. For genomic DNA, single-crossover or a replicating plasmid no pre-treatment is needed.

1) Thaw competent cells (if needed) at 37°C and transfer cells to a 15 ml polypropylene tube.

2) Add DNA (20 ul of plasmid digest, 10 ul of circularised plasmid or genomic DNA) and incubate at 37°C with shaking at 200 rpm for 1 hour.

3) Add 700 ul LB medium and continue growth for 1.5-2 hours depending on resistance marker for selection (e.g. 1.5 hours for erythromycin, 2 hours for chloramphenicol)

4) Plate 20-200 ul onto LB agar, grow for 18-24 hours at 37°C.

Media

Minimal salts solution (5x)

Ammonium sulphate, 2 g; potassium hydrogen phosphate, 14.8 g; potassium dihydrogen phosphate, 5.4 g; sodium citrate, 1.9 g; magnesium sulphate heptahydrate, 0.2 g. Dissolve in 150 ml deionised water and adjust to pH 7.0 with hydrochloric acid/sodium hydroxide. Adjust volume to 200 ml and autoclave.

Minimal growth medium

Per 50 ml: minimal salts solution, 10 ml; glucose (50% (w/v)), 0.5 ml; casamino acids (2% (w/v)), 0.5 ml; tryptophan (10 mg/ml), 0.1 ml; iron ammonium citrate (2.2 mg/ml), 0.05 ml, deionised water, 39 ml. Prepare freshly and filter sterilise.

Starvation medium

Per 50 ml: minimal salts solution, 10 ml; glucose (50% (w/v)), 0.5 ml; deionised water, 39.5 ml. Filter sterilise before use.