Team:Calgary/Notebook/ProtocolManual/Bsubtilis
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B. subtilis Protocols
B. subtilis preparation and transformation (Zhang & Zhang, 2010)
- 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.
- Cultivate cells overnight at 37°C, shaking at 200rpm.
- Dilute culture to an OD of 1.0 (A600) in fresh LB containing 1% (w/v) xylose.
- Grow cells for 2 hours at 37°C, shaking at 200rpm.
- Divide into aliquots and store at -80°C with 10% (w/v) glycerol OR proceed with transformation.
- Add 1μL PCR product to 100μL supercompetent cells in a plastic tube.
- Cultivate cells for 1.5 hours at 37°C, shaking at 200rpm.
- Spread on LB plates (with appropriate antibiotic selection).
- 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.
- 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.
- Cultivate cells overnight at 37°C, shaking at 200rpm.
- *Take initial absorbance reading at A600 and calculate difference to 1.0; dilute to 1.0 with fresh LB using standard curve.
- *Add 20% xylose for 2% final xylose concentration.
- Grow cells for 2 hours at 37°C, shaking at 200rpm.
- Divide into aliquots and store at -80°C with *25% (w/v) glycerol OR proceed with transformation.
- *Calculate volume for 400ng DNA and add to 100μL supercompetent cells in a plastic tube.
- *Cultivate cells for >2 hours at 37°C without shaking.
- Spread on LB plates (with appropriate antibiotic selection).
- 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.
Bacterial smear
- Glass microscope slides
- Draw a one inch circle on the back of a glass microscope slide
- Pipet 50 μL of distilled water in the circle and use a sterile inocculation loop to transfer a colony of bacteria into this water
- Fix bacteria on to the slide by running it through a flame a few times
- Allow the smear to dry before performing any stains
Gram stain
- Crystal violet
- Lugol's iodine
- Safrinin
- Flood bacterial smear with Crystal Violet for one minute
- Rinse with distilled water
- Flood with Lugol's Iodine for one minute
- Rinse with distilled water
- 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
- Counter-stain with Safrinin for one minute
- Rinse one last time with distilled water and pat dry with Kim-wipes
Gram-negative cells will stain pink or red, and Gram-positive cells will stain purple or blue.
Spore stain
- Malachite green
- Safrinin
- 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
- Place slides onto a hot plate at 150 °C
- Place a piece of paper towel over the smear to allow for complete flooding and steaming with Malachite Green
- Continuously flood the paper towl to prevent drying for five minutes
- Rinse with distilled water
- Counter-stain with Safrinin for one minute
- Rinse one last time with distilled water and pat dry with Kim-wipes
Cells will stain pink and spores will stain green.