Team:Fatih Turkey/bsubtilis

BACILLUS SUBTILIS (competent ve transformation)

Media Preparation

10X Medium A base:

● Yeast extract 10g

● Casamino acids(pepton from casein/ triptone ) 2g

● Distilled water to 900mL

● Autoclave, then add :

● 50% glucose, filter sterilized 100mL (50 g/100 mL)(%50 glikozu distile suyle birlikte bir flaskın içinde bunsen burnerda ısıtarak çöz)

10X Bacillus salts:

● (NH4)2SO4 20g

● Anhydrous K2HPO4 139.7g

● KH2PO4 60g

● Tri-sodium citrate 10g

● MgSO4•7H2O 2g

● SDW(sterile distile water) to 1000mL

● Then, autoclav

Medium A

● Sterile water 81mL

● 10X Medium A base 10mL

● 10X Bacillus salts 9mL

● L-Tryptophan (11mg/mL) 0.1mL (filter sterilized)

● Then, filter sterilized

Medium B

● Medium A 10mL

● 50mM CaCl2•2H2O 0.1mL (filter sterilized) (147 g/mol)

● 250nM MgCl2•6H2O 0.1mL (filter sterilized) (203.3 g/mol)(hazırlanışı için notlar kısmızı oku)

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 37ºC (plate been kept on the bench for several days would be better)
2. Inoculate about 12mL of medium 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 37ºC with vigorous shaking. Read the OD650 every 20min (never keep the solution you used for measuring!)
4. Plot log(OD650) in function of time. After a brief lag, you should observe a exponential increase. After awhile, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t0 (3 on the graph). It should take about 100min and the OD should be between 0.35 and 0.55.

 

1. At t0, incubate for 90 minutes at 37ºC with vigorous shaking.
2. Transfer 0.05mL of this culture into 0.45mL of pre-warmed Medium B in an Eppendorf tube. You have to prepare one tube for each transformation, plus an extra tube for a DNA-less control.
3. Incubate the diluted cultures at 37ºC with shaking for 90min. At this moment, the cells are HIGHLY COMPETENT.
4. To check for competency, you can look at cells under the microscope; competent cells are very motile.

Transforming

1. Spin Eppendorf tubes containing cells. Remove 400µL of liquid to keep only 100µL of the culture (to concentrate cells). Re-suspend the cell pellet in the remaining culture.
2. To transform from competent glycerol stocks, firstly thaw on ice and then spin the tube at about 1600rpm for 20min, remove the supernatant (glycerol), and add 100µL of pre-warmed medium B.
3. Mix the cells thoroughly. (pipeting slowly)
4. Add 0.6µg of DNA to the competent cells. (yarın belli oacak)
5. Incubate for 30min at 37ºC with shaking.
6. Plate 100µL of transformed cells onto selective agar.

Glycerol Stocks

1. To freeze competent Bacillus cells, spin down(8ooo rpm, 5 min) the fresh competent cells to obtain a pellet.
2. Remove all supernatant. (remove 450µLsupernatant with pipette)
3. Re-suspend cells in 500µL 60% glycerol. (slowly)
4. Add into liquid nitrogen
5. Freeze tubes at -80ºC.