BL21(AI) 感受态细胞(化转)/ BL21(AI) c

BL21(AI) chemically Competent Cells

 

BL21(AI) Chemically Competent Cell Components

BL21(AI)锛?nbsp;                                                  100μl/tube

pUC19 (control vector锛?0pg/μl):                    10μl

Storage at                             -80鈩冿紙6month锛?/span>

 

BL21(AI) Chemically Competent Cell Genotype

F- ompT hsdSB(rB-mB-) gal dcm araB::T7RNAP-tetA

 

BL21(AI) Chemically Competent Cell Description

BL21 (AI) is a strain of Escherichia coli B/r (E.coli B/r). BL21 (AI) was derived from BL21 strain, which was a defective strain of Lon protease and extracellular protease OMPT. The deletion of these two enzymes effectively prevented the degradation of heterologous protein in Escherichia coli. The addition of L- Arabia sugar in the medium can induce the expression of T7RNA polymerase in the downstream of araBAD promoter to promote the expression of the target protein. Adding glucose to the medium could inhibit the expression of T7RNA polymerase in the downstream of araBAD promoter and then inhibit the expression of the target protein. BL21 (AI) receptive cells are suitable for any expression vector based on the promoter of T7, which can be used to express the high level of recombinant protein. Because strains can efficiently regulate the level of T7 RNA polymerase in vivo, BL21 (AI) competent cells can express toxic or inhibitory growth proteins to other BL21 cells. The yield of common recombinant protein in BL21 (AI) strain is the same as that of other BL21 strains. The yield of most toxic protein in BL21 (AI) strain is higher than that of BL21 (DE3) pLysS strain or BL21 (DE3) strain. The BL21 (AI) receptive cells were made by special technology, and the conversion efficiency of pUC19 plasmid was as high as 108cfu/ mu g DNA.

 

BL21(AI) Chemically Competent Cell Transformation Protocol
1. Please store BL21(AI) Chemically Competent Cell in -80°C for late use.
Take it then insert the tube in the ice immediately for 5 minutes.
2. Add your plasmid DNA (vector) to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.Place the mixture on ice for 25 minutes,Do not mix.
3. Place it in the 42°C water for 45 seconds,then immediately transfer the tube back into ice for 2min. Do not mix.
4. Add 700ul LB or 2YT liquid nutrient medium (No antibiotic). then put on the Constant temperature shaker (200 rpm) for 1 hours at 37°C.
5.Pellet the mixture by centrifugation at 5000rpm/min for 1min. Pour off about 100ul of cell supernatant, then resuspend the cell pellet in the remaining medium by gently vortexing the tube. Spread the cell on the LB or 2YT Resistance plates by antibiotic, then inverted and Incubate overnight at 37°C.(bule white selection should place the plate at 37°C for 14 hours or more).

Caution锛?/span>

1. Brian Caliendo (Voigt lab)reported that PCP20 plasmids are difficult to be transformed into this receptive cell, and the transformation of pCP20 into other strains is normal, but the cause is unknown.

2. Without glucose, the downstream expression level of araBAD promoter in BL21 (AI) cells is still very low. After glucose addition, it can further reduce the expression level of background protein.

 

Sample Induction Protocol (for reference only)

1. Pick 3-4 transformants for overnight culture in 5 mL LB medium containing antibiotic to select for your expression plasmid. Grow overnight at 37°C with shaking until the OD600 reaches 0.6-1.0.

2.Use the overnight cultures to inoculate fresh LB medium containing antibiotic to an OD600 of 0.05-0.1 (~1:20 dilution of the overnight culture). This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density. Use a volume appropriate for taking time points, if desired.

3.Use the remainder of each overnight culture to create glycerol stocks. Once you have identified the clone that best expresses your protein, you can use the glycerol stock to perform additional expression experiments.

4.Grow the cultures until they reach mid-log phase (OD600 ~0.4; 2 to 3 hours).

5.Induce the cultures (see below), and culture for an additional 2-3 hours. You may also take time points to analyze for optimal expression of your protein.

For T7 expression vector containing the lacI gene (e.g. Invitrogen’s pET vectors), induce by adding L-arabinose to a final concentration of 0.2% AND IPTG to a final concentration of 1 mM.

For T7 expression vector with no lacI gene (e.g. Invitrogen’s pCR®T7 vectors), induce by adding L-arabinose to a final concentration of 0.2%. Culture for an additional 2-3 hours.

6.Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000×g for 10 minutes at 4鈩?

7. Remove the supernatant and store the cell pellet at -20鈩?(storage at lower temperatures is also acceptable).  IPTG

Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside / thiogalactopyranoside) by dissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.

 

Caution

1., The competent cells are best to melt slowly in the ice, and insert the target DNA into the ice for 8 minutes. They cannot be placed in the ice for too long. Long time storage will reduce the conversion efficiency.

2. Should be softly manipulate when mixed with plasmids.

3. Transformation of high concentration plasmids could reduce the amount of bacteria that were eventually used for coated plates.

4. When induction, the concentration of IPTG is optional (0.1-2mM).

5. In order to obtain the required amount of protein, the optimum induction time, temperature, and IPTG concentration should be optimized.

 

Suggestion Expression Condition锛?/span>

1. Protein expression was performed using T7 promoter (high or low copies).

2. When other BL21 strains were used to express protein, the inhibitory effects of bacterial growth were observed.

3. Expressed a known toxic protein.