Transformation of Bacillus subtilis

Materials required

1. Bacillus subtilis 168
2. 10 µl of transformation DNA
3. Pipettes
4. 2X 15 ml falcon tubes
5. 2X 50 ml falcon tubes
6. Eppendorf tubes
7. Agar plates containing the appropriate antibiotic
8. Water bath
9. Centrifuge
10. SMM medium (1 liter)
    • 2.0 g of ammonium sulphate
    • 14.0g of dipotassium hydrogen phosphate
    • 6.0g of potassium dihydrogen phosphate
    • 1.0g of sodium citrate dehydrate
    • 0.2g of magnesium sulphate
    • Top up the rest of the medium with water
11. MM competence medium
    • 10 ml of SMM medium
    • 125 µl of Solution E (40% glucose)
    • 100 µl of Tryptophane solution (at a concentration of 2mg/ml) –
    • 60 µl of Solution F (1M MgSO4)
    • 10 µl of 20% Casamino acids
    • 5 µl of 0.22% Fe-NH4-citrate
12. Starvation medium
    • 10 ml of SMM medium
    • 125 µl of Solution E (40% glucose)
    • 60 µl of Solution F (1M MgSO4)

Protocol

This protocol will stretch for 2 days and aseptic technique have to be applied for all steps.

Protocol for Day 1

1. Inoculate a single colony of Bacillus subtilis 168 into a 15 ml falcon tube containing 10 ml of MM competence media.
2. For control transfer 10 ml of MM competence media without the bacteria.
3. Incubate overnight in a shaking incubator at 37ºC.

Protocol for Day 2

1. Transfer 0.6 ml of the overnight culture into 50 ml falcon tube containing 10 ml of MM competence medium.
2. Incubate the tubes for 3 hours at 37ºC.
3. Warm up the starvation medium to 37ºC in a water bath.
4. Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 hours at 37ºC.
5. Transfer 1X 0.4 ml of the medium not containing B. subtilis into one Eppendorf tube.
   • Add 10 µl of water into the tube.
6. Transfer 2X 0.4 ml of the medium containing B. subtilis into two Eppendorf tubes.
   • Add 10 µl of DNA into one tube.
   • Add 10 µl of water into one tube.
7. Incubate the samples for 1 hour at 37ºC in the shaking incubator.
   • The Eppendorf tubes have to be aerated, therefore incubate the tubes on their side.
8. Centrifuge the Eppendorf tubes at 13,000 rpm for 2 minutes.
9. Discard 0.3 ml of supernatant from each Eppendorf tubes.
10. Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
11. Incubate the plates overnight at 37ºC.

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