Team:Cambridge/Protocols/TransformationofB.subtilis

Transformation of Bacillus subtilis

A technique used to introduce foreign DNA into competent Bacillus cells.

Media Preparation

10X Medium A base:

• Yeast extract 10g
• Casamino acids 2g
• Distilled water to 900mL
• Autoclave then add:
• 50% glucose, filter sterilized 100mL

10X Bacillus salts:"

• (NH₄)₂SO₄.3H₂O 20g
• K₂HPO₄.3H₂O 183g
• KH₂PO₄ 60g
• Tri-sodium citrate 10g
• MgSO₄.7H₂O 2g
• SDW to 1000mL

Medium A

• Sterile water 81mL
• 10X Medium A base 10mL
• 10X Bacillus salts 9mL
• L-Tryptophan (11mg/mL) 0.1mL

Medium B

• Medium A 10mL
• 50mM CaCl₂.2H₂O 0.1mL
• 250nM MgCl₂.6H₂O 0.1mL

Important

• Autoclave Medium A base before adding glucose, and autoclave Bacillus salts.
• Store aliquots of 10X Medium A base 10mL and 10X Bacillus salts 9mL and keep them in the fridge, never use them twice to avoid contamination

Protocols

Making Bacillus competent

1. Grow one blank plate of Bacillus subtilis (or several if you want to transform different strains) for 20 hours at 37^oC (plate kept on the bench for several days would be better)
2. Inoculate about 12ml of Medium A with several colonies. Mix the contents of the tube. Check with OD₆₅₀. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8ml of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture.
3. Incubate at 37^oC with vigorous shaking. Read the OD₆₅₀ every 20 minutes (always dispose of the sample used for measuring)
4. Plot log(OD₆₅₀) as a function of time. After a brief lag, you should observe an exponential increase. After a while, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t₀. This should take about 100 minutes and the OD should be between 0.7 and 1.0.
5. At t₀ incubate for 90 minutes at 37^oC with vigorous shaking.
6. Transfer 0.05ml of this culture into 0.45ml of pre-warmed Medium B in an Eppendorf tube. One tube needs to be prepared for each transformation, plus one extra for a DNA-less control.
7. Incubate the diluted cultures at 37^oC with shaking for 90 minutes. At this moment, the cells are HIGHLY COMPETENT.
8. To check for competency, you can look at cells under the microscope; competent cells are very mobile.

Transforming

1. Spin Eppendorf tubes containing cells. Discard 400µl of the liquid medium (leaving 100µl - this will concentrate the cells). Resuspend the pellet in the remaining culture.
2. To transform from competent glycerol stocks, spin the tube at about 11000rpm for 20 minutes, remove the supernatent (glycerol), and add 100µl of pre-warmed medium B
3. Mix the cells thoroughly
4. Add 0.6µg of DNA to the competent cells
5. Incubate for 30min at 37^oC with shaking
6. Plate 100µl of transformed cells onto selective agar

Glycerol stocks

1. To freeze competent Bacillus cells, spin down the fresh competent cells to obtain a pellet
2. Remove all supernatant
3. Re-suspend cells in 500µl 60% glycerol
4. Freeze tubes at -80^oC

Safety

All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved for decontamination purposes.