Bacillus sub Expression

orage of recombinant bacillus

Short-term storage (about 1 week)

Pick a single colony and spread on a MTNm plate.
Place the plate in an incubator at 30°C overnight.
Seal the plate and store it at room temperature (about 20°C).

Do not store the plates in a refrigerator.

Long-term storage (1 month or longer)

Pick a single colony and inoculate into 2SYNm medium. Culture overnight on a shaker.

Transfer the broth to a freezing vial and add an equal amount of LB medium containing 40% glycerol.
Store at -80°C. 

B. subtilis Competent Cell Preparation and Transformation

Very Important:

ampicillin selection is for E. coli only. The amp gene is not expressed in Bacillus or other Gram-positive organisms. Use erythoromycin (1 μg/ml) with pLIKE-rep or chloramphenicol (5 μg/ml) with pLIKE-int for selection in B. subtilis

For pHCMC04: Use 5 µg/mL Chloramphenicol for E coli and Bacillus, use ampicillin in E coli

Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975. Transformation and transfection in lysogenic strains of Bacillus subtilis: Evidence for selective induction of prophage in competent cells. J. Bacteriol. 121:296-304. Zahler, S. A. (personal communication)

Competence development in B. subtilis is one of several stationary phase processes triggered by a nutritional downshift. Since the pioneering work of Anagnostopolous and Spizizen, a number of protocols for preparing competent B. subtilis cells have appeared. The following method, modified by Ron Yasbin from protocols developed in the Frank Young lab at Rochester, was used routinely in Stan Zahler’s wonderful bacterial genetics course at Cornell. This protocol assumes that you use a spectrophotometer that accepts 16×125 mm test tubes. If your spectrophotometer, like mine, works only with cuvettes, simply increase the culture volume to 10 or 20 ml in a 250-ml Erlenmeyer flask.

1. Streak recipient strain on one-half of a Tryptose Blood Agar Base plate. Incubate overnight (18 hr) at 37°C.

2. Inoculate a few colonies into 4.5 ml of Medium A in a 16×125 mm test tube that lacks visible scratches. Mix the contents of the tube thoroughly. Read its optical density at 650 nm in the spectrophotometer. Adjust the OD 650 to be 0.1-0.2, maintaining the volume at 4.5 ml.

3. Incubate at 37°C with vigorous aeration. Read the OD 650 every 20 min, plotting OD 650 against time on semi-log paper. After a brief lag, the OD should increase logarithmically—that is, they should fall on a straight line. Note the point at the culture leaves log growth—the graph points fall below the straight line. In B. subtilis genetics, this point is known as t 0 . It should take 60-90 minutes of incubation and occur at OD 650 =0.4-0.6.

4. Continue incubation for 90 minutes after the cessation of log growth (t 90 ). Transfer 0.05 ml of this culture into 0.45 ml of pre-warmed Medium B in a 16×125 mm test tube. Set up one tube for each transformation you intend to perform, plus an extra for a DNA-less control.

5. Incubate the diluted cultures at 37°C with vigorous aeration for 90 min. At this point, the cultures should be highly competent.

6. Add 1 μg of DNA to the competent cells and incubate at 37°C with aeration for 30 minutes.

7. Plate aliquots of the transformed cells onto selective agar.

Medium A
Sterile water: 81 ml
10× Medium A base: 10 ml
10× Bacillus salts:  9 ml

autoclave

Medium B
Medium A: 10 ml
50 mM CaCl2 ·2H2O:  0.1 ml
250 mM MgCl2 ·6H2O:  0.1 ml

10× Medium A base:
Yeast extract:  10 g
Casamino acids:  2 g
Distilled water to 900 ml
50% glucose: 100 ml

Autoclave.

10× Bacillus salts

(NH4)2SO4:  20 g
K2HPO4·3H2O:  183 g
KH2PO4:  60 g
Na + citrate:  10 g
MgSO4·7H2O:  2 g
Water  to 1000 ml

Transformation Protocol 2

Media composition

Starvation medium 1 (SM1) contains

• 0.2% ammonium sulfate
• 1.4% dipotassium hydrogen phosphate
• 0.6% potassium dihydrogen phosphate
• 0.07% sodium citrate
• 0.5% glucose
• 0.02% magnesium sulfate heptahydrate
• 0.2% yeast extract

• 0.025% casamino acids.
  

Mix all components of SM1 medium and autoclave together.

Starvation medium 2 (SM2) is less rich in nutrients and includes

• 0.2% ammonium sulfate,
• 1.4% dipotassium hydrogen phosphate,
• 0.6% potassium dihydrogen phosphate,
• 0.07% sodium citrate,
• 0.5% glucose,
• 0.08% magnesium sulfate heptahydrate,
• 0.1% yeast

extract,

• 0.01% casamino acids
• 0.05% calcium chloride
  

Mix all components of SM2 medium  and autoclave together.

Histidine solution was sterilized by filtration using 0.2-μm filters

Preparation of competent cells

Sprea B. subtilis DB104 cells  a LB agar plate without antibiotics (37 °C; for 9 h).

Inoculate antibiotic-free SM1 media  by transferring a single colony and subsequent cultivation (37 °C; 250 rpm for 14–16 h).  

Dilute vernight culture  in SM1 medium by adjusting optical density at 600 nm (OD 600 ) to 0.5 (approximately 2.9×10 7 cells/ml) in a volume of 10 ml and incubated (37 °C; 200 rpm for 3 h).

The cell culture volume is doubled by addition of SM2 medium and subsequently supplemented with varied histidine concentrations (final concentration 0, 10, 50, 200, 500, and 1,000 μg/ml; total volume 20 ml) and incubate (37 °C; 300 rpm for 2 h). After the treatment, B. subtilis DB104 cells are competent for approximately 1 h. Histidine concentration of 200 μg/ml (final concentration) is used in final protocol. 

Transformation procedure

Five hundred microliters of the competent cells is mixed with varied amounts of plasmid DNA (pHY300Car—2, 5, 10, 20, 40, 100, 250, 500, 1,000 ng) and incubate (37 °C; 200 rpm for 30 min). In order to recover the cells, 300 μl of fresh LB medium is supplemented and competent B. subtilis DB104 are additionally incubated (37 °C; 200 rpm for 30 min). Two hundred microliters of B. subtilis DB104 cell suspension is subsequently spread on LB agar plates with selective antibiotic (tetracycline, final concentration 15 μg/ml).

Transformation Protocol 3

An overnight culture was grown in minimal medium with 2.5 μg/ml erythromycin at 37°C, with continuous shaking at 300 rpm. After 100-fold dilution of the overnight culture in minimal medium, incubation was continued, and the optical density at 600 nm (OD600) was monitored. Two hours after the transition from exponential to stationary-phase growth, 1 μg of DNA was added to 0.5 ml of competent cells. Samples were kept at 37°C with shaking. After 20 min, 0.3 ml of TY medium was added, and growth was continued for another 30 min, after which the cells were plated on selective TY-agar plates.

Transformation Protocol 4

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 20g
• Anhydrous K2HPO4 139.7g
• 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

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.

OD650 of B.subtilis

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, 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.
4. Add 0.6µg of DNA to the competent cells.
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 the fresh competent cells to obtain a pellet.
2. Remove all supernatant.
3. Re-suspend cells in 500µL 60% glycerol.
4. Freeze tubes at -80ºC.

Results

Transformation with episomal vectors

• Selectable on Chloramphenicol 5µg/mL

Transformation with integration vectors

• Selectable on Spectinomycin 50µg/mL