Competent preparation: preparation of competent cells of Bacillus subtilis

October 11, 2022

The preparation of Bacillus subtilis competent cells can be: (1) used to establish a Bacillus subtilis transformation system; (2) for its genetic modification; (3) for improving the production performance of Bacillus subtilis.

experimental method
  • Chemical method
  • Chemical improvement method
Experimental Materials
Bacillus subtilis 168
Reagents, kits
SPI medium EGTA SPII medium sodium citrate EGTA sodium hydroxide KH2PO4 K2HPO4 MgCl2 MgSO4 glucose Yeast Extract (NH4) 2SO4
Instruments, consumables
Incubator Petri dish Conical flask UV spectrophotometer Inoculation ring Centrifuge tube
Experimental procedure
First, reagent preparation

1. SP salt

0.2% (NH 4 ) 2 SO 4 , 1.4% K 2 HPO 4 , 0.6% KH 2 PO 4 , 0.02% MgSO 4 ·7H 2 O, 0.1% sodium citrate.
2. CAYE (100×)

2% Casamino acid, 10% yeast extract.
3. SPI medium

The SP salt solution was added to a 1% by volume solution of 50% glucose solution, 1% by volume of 100 x CAYE solution.

4. SPII medium

SPI medium was added with 1% by volume of 50 mmol/L CaCl 2 solution and 1 % by volume of 250 mmol/L MgCl 2 solution.
5. SP-A Salts Solution (500 ml)

(1) 0.4% (NH 4 ) 2 SO 4 : 2 g

(2) 2.8% K 2 HPO 4 ·3H 2 O : 14 g

(3) 1.2% KH 2 PO 4 : 6 g

(4) 0.2% Trisodium Citrate Dihydrate : 1g

(5) Sterilization at 121 ° C for 20 min.
6. SP-B Salts Solution (500 ml)

(1) 0.04% MgSO 4 ·7H2O: 0.2 g

(2) Sterilization at 121 ° C for 20 min.
7. 100×CAYE Solution (100 ml)
(1) 2% Casamino acid : 2 g

(2) 10% Yeast Extract: 10 g

(3) Sterilization at 121 ° C for 20 min.
8. SPI Medium (20 mL)

SP-A Salts Solution: 9.8 mL

SP-B Salts Solution: 9.8 mL

(1% V) Glucose (50% w/v, sterilized at 115 ° C for 20 min): 200 μL

(1%V) 100×CAYE: 200 μL
10. SPII Medium (6 mL)

SPI Medium: 5.88mL

(1% V) 50 mM CaCl 2 : 60 μL

(1% V) 250 mM MgCl 2 : 60 μL
11. 100×EGTA Solution

10mmol / L EGTA solution, a small amount of NaOH should be added to pH 8.0 when dissolved.

Second, the experimental steps

1. Prepare fresh 168 monoclonal plates, take a full-circle Bacillus subtilis glycerol LB plate, and incubate in a 37 °C incubator for 12 h.
2. The colony was picked up in 3 ml of LB medium the night before the transformation, and cultured overnight at 37 ° C, 250 r/min.
3. The next morning, 160 μl of the culture medium was transferred to 8 ml of SPI medium, and cultured at 37 ° C, 250 r/min until the end of logarithmic growth (168 about 4-5 h).
4. Take 0.2 ml of the culture medium grown to the end of the logarithmic phase to 2 ml of SPII medium, and incubate at 37 °C for 100 minutes at 100 r/min.
5. Add 20 μl of 10 mmol/L EGTA to the cells of the above SPII medium, and incubate at 37 ° C, 100 r / min for 10 minutes.
6. Dispense the above treated liquid into 0.5 ml each tube, add 5 μl of DNA each (the amount of DNA should not be too high, no more than 5 μg), and then incubate at 37 °C, 250 r/min for 90 minutes. The bacterial solution was applied to screen the plate.
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Precautions
1. Be aware of the cleanliness and sterility of the instrument.

2. 8ml SPI medium should be placed in a 50 ml centrifuge tube to ensure the growth of the cells, do not use glass tubes.

3. Note that the OD value must be measured and the cells must be grown until the late log phase.

4. Ensure the concentration of the bacteria to increase the conversion rate.

Yeast Extract

Yeast Extract,Autolyzed Yeast Extract,Yeast Extract Powder,Hydrolyzed Yeast Extract

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