What is the difference between BL21 and BL21 DE3?
The DE3 designation means that respective strains contain the λDE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. Note that BL21 does not carry the gene for T7 RNA polymerase and thus is only suitable for expression from promoters recognized by the E.
Why we use BL21 for protein expression?
The BL21(DE3)pLysS competent cells provide tighter control of protein expression for expression of toxic proteins and are resistant to chloramphenicol. When used with the CE6 bacteriophage, the BL21 cells provide the tightest control of protein expression (see BL21(DE3) Strains and Protein Toxicity).
Can I extract plasmid from BL21 cells?
BL21 cells are not designed for plasmid purification but protein expression. BL21 lacks in some proteases but has the endonucleases present. You can make mini-preps and try to isolate the plasmid, but there is a risk that your plasmid may be digested.
How do I make BL21 DE3 competent?
- (For C2527H) Thaw a tube of BL21(DE3) Competent E.
- Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture.
- Place the mixture on ice for 30 minutes.
- Heat shock at exactly 42°C for exactly 10 seconds.
- Place on ice for 5 minutes.
- Pipette 950 µl of room temperature SOC into the mixture.
What are BL21 DE3 cells?
BL21(DE3) is a chemically competent E. coli cell suitable for transformation and high level protein expression using a T7 RNA polymerase-IPTG induction system.
What are BL21 cells?
BL21 competent cells are an all-purpose strain for high-level protein expression and easy induction. The basic BL21 strain does not contain the T7 RNA polymerase gene and can be used with non-T7 RNA polymerase protein expression systems. BL21 has the tightest control of protein expression for extremely toxic proteins.
What is BL21 DE3 cells?
How do you transform in BL21?
BL21 Chemically Competent Cells are transformed in 40 μL reactions. Cells are packaged with sufficient volumes for 1, 2, or 4 reactions per tube. Transformation is performed by heat shock at 42 °C, followed by incubation on ice.
What does BL21 DE3 mean?
BL21(DE3) is an E. coli B strain and does not contain the lon protease. It is also deficient in the outer membrane protease OmpT. The lack of these two key proteases reduces degradation of heterologous proteins expressed in the cells.
What is E. coli BL21 DE3?
E. coli BL21(DE3), a derivative of BL21, is probably the most widely used in high-level expression of recombinant proteins, and it harbors a prophage DE3 derived from a bacteriophage λ, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter.
Why were BL21 Gold cells used?
‡ BL21-Gold(DE3) is an all-purpose strain for high-level protein expression and easy induction. The BL21- Gold(DE3)pLysS strain provides tighter control of protein expression for expression of toxic proteins. When used with the CE6 bacteriophage, the BL21-Gold cells provide the tightest control of protein expression.
How much DNA do you need for BL21 transformation?
Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA.