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

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

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 autoclave.
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 50-10,000 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 4°C.
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.

Bacterial smear

• Glass microscope slides

1. Draw a one inch circle on the back of a glass microscope slide
2. Pipet 50 μL of distilled water in the circle and use a sterile inocculation loop to transfer a colony of bacteria into this water
3. Fix bacteria on to the slide by running it through a flame a few times
4. Allow the smear to dry before performing any stains

Gram stain

• Crystal violet
• Lugol's iodine
• Safrinin

1. Flood bacterial smear with Crystal Violet for one minute
2. Rinse with distilled water
3. Flood with Lugol's Iodine for one minute
4. Rinse with distilled water
5. Decolourize smear using alternating applications of 95% ethanol for ten second and distilled water for ten seconds until the water running off of the slide runs clear
6. Counter-stain with Safrinin for one minute
7. Rinse one last time with distilled water and pat dry with Kim-wipes

Gram-negative cells will stain purple or blue, and Gram-positive cells will stain pink or red.

Spore stain

• Malachite green
• Safrinin

1. Collect the spores for the smear from the suspended spores by centrifuging them for two minutes at 14,000 rpm, and use an inocculation loop to collect the spores
2. Place slides onto a hot plate at 150 °C
3. Place a piece of paper towel over the smear to allow for complete flooding and steaming with Malachite Green
4. Continuously flood the paper towl to prevent drying for five minutes
5. Rinse with distilled water
6. Counter-stain with Safrinin for one minute
7. Rinse one last time with distilled water and pat dry with Kim-wipes

Cells will stain pink and spores will stain green.