Team:Calgary/Notebook/ProtocolManual/Bsubtilis

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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 an OD of 1.0 (A600) 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)

The changes to the original protocol are indicated by asterisks (*), and supported by figures where appropriate.

  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.

B. subtilis colony PCR

  • Pick a colony and resuspend in 50μL of TE buffer.
  • Heat for 5 minutes at 100°C.
  • Spin down for 30 seconds and use 5μL for the PCR.

    • Making 2x SG Media for sporulation of B. subtilis

  • 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.
  • Adjust the pH to 7.0 and autoclave.
  • 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

  • Inoculate 10-50mL of LB medium with cells from a fresh colony of B. subtilis
  • Grow for 6-8 hours at 37°C with shaking at 200rpm.
  • Dilute 1/200 into 50-10,000 ml 2xSG medium in an appropriate vessel and grow at 37°C with shaking at 200rpm.
  • After 2-3 days, over 90% of the population should be spores. Pellet cells at 9000rpm for 20minutes at 4°C.
  • Wash spores with ice-cold water 8-10 times.
  • Store at 4°C with weekly changes of water OR at -20°C for long-term storage.

    • Retrieved from "http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/Bsubtilis"