B. subtilis Protocols

B. subtilis preparation and transformation (Zhang & Zhang, 2010)

1. Inoculate B. subtilis SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.
2. Cultivate cells overnight at 37°C, shaking at 200rpm.
3. Dilute culture to A600 at 1.0 in fresh LB containing 1% (w/v) xylose.
4. Grow cells for 2 hours at 37°C, shaking at 200rpm.
5. Divide into aliquots and store at -80°C with 10% (w/v) glycerol OR proceed with transformation.
6. Add 1μL PCR product to 100μL supercompetent cells in a plastic tube.
7. Cultivate cells for 1.5 hours at 37°C, shaking at 200rpm.
8. Spread on LB plates (with appropriate antibiotic selection).
9. Incubate plates at 37°C for 20 hours.

B. subtilis preparation and transformation (modified from Zhang & Zhang, 2010)
1. Inoculate B. subtilis SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.
2. Cultivate cells overnight at 37°C, shaking at 200rpm.
3. Take initial absorbance reading at A600 and calculate difference to 1.0; dilute to 1.0 with fresh LB using standard curve.
4. Add 20% xylose for 2% final xylose concentration.
5. Grow cells for 2 hours at 37°C, shaking at 200rpm.
6. Divide into aliquots and store at -80°C with 25% (w/v) glycerol OR proceed with transformation.
7. Calculate volume for 400ng DNA and add to 100μL supercompetent cells in a plastic tube.
8. Cultivate cells for >2 hours at 37°C without shaking.
9. Spread on LB plates (with appropriate antibiotic selection).
10. Incubate plates at 37°C for 20 hours.

Making 2x SG Media for sporulation of B. subtilis
1. Measure 16.0g Difco nutrient broth, 2.0g KCl, and 0.5g MgSO4∙7H2O. Add to large bottle and add distilled water for a 1L solution.
2. Adjust the pH to 7.0 and autoclate.
3. After cooling, add the following sterile solutions: 1mL of 1M Ca(NO3)2, 1mL of 0.1M MnCl2∙4H2O, 1mL of 1mM FeSO4, and 2mL of 50% (w/v) glucose.

Generating and harvesting B. subtilis spores
1. Inoculate 10-50mL of LB medium with cells from a fresh colony of B. subtilis
2. Grow for 6-8 hours at 37°C with shaking at 200rpm.
3. Dilute 1/200 into 50-10000 ml 2xSG medium in an appropriate vessel and grow at 37°C with shaking at 200rpm.
4. After 2-3 days, over 90% of the population should be spores. Pellet cells at 9000rpm for 20minutes at low temperatures.
5. Wash spores with ice-cold water 8-10 times.
6. Store at 4°C with weekly changes of water OR at -20°C for long-term storage.