What is the difference between BL21 and BL21 DE3?

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.

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.

What is BL21 DE3 pLysS?

BL21(DE3) is an expression strain suitable for high level induction and expression of genes from any T7 promoter-based expression vector. T7 RNA polymerase expression is induced by the addition of 1mM IPTG to the culture.

How does pLysS work?

pLysS is a plasmid that contains the T7 lysozyme gene (LysS). The T7 lysozyme binds to T7 RNA polymerase causing inhibition until induction by the addition of IPTG. When IPTG is added, the amount of T7 RNA polymerase increases and over- comes the inhibition by LysS.

Does BL21 have T7 polymerase?

It is acknowledged that the pET expression system is a powerful tool for the production of recombinant proteins. BL21 (DE3) carries an inducible T7 RNA polymerase-dependent pET expression system that allows for the simple manipulation and tuning of protein production levels.

Why do 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).

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.

How do you transform a plasmid into BL21?

Transformation Protocol for BL21(DE3) Competent Cells (C2527)

1. (For C2527H) Thaw a tube of BL21(DE3) Competent E.
2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture.
3. Place the mixture on ice for 30 minutes.
4. Heat shock at exactly 42°C for exactly 10 seconds.
5. Place on ice for 5 minutes.

Can you isolate plasmid from BL21?

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.

What is the difference between DH5 alpha and BL21?

The key difference between BL21 and DH5 Alpha is that BL21 is a protease deficient genetically engineered competent E. coli cell used primarily for protein expression, while DH5 Alpha is a genetically engineered competent E. coli cell with recA1 mutation used primarily for plasmid transformation.

Does BL21 have T7?

BL21 (DE3) carries an inducible T7 RNA polymerase-dependent pET expression system that allows for the simple manipulation and tuning of protein production levels.

Why we use BL21 for protein expression?

Why BL21 (DE3) plyss cells?

BL21 (DE3)pLysS cells are ideal for use with T7 promoter-based expression systems (e.g., pRSET, pCR™T7, and pET). BL21 (DE3)pLysS E. coli carry both the DE3 lysogen and the plasmid pLysS. pLysS constitutively expresses low levels of T7 lysozyme, which reduces basal expression of recombinant genes by inhibiting basal levels of T7 RNA polymerase.

What is BL21 DE3 competent?

The BL21(DE3) competent cells are an all-purpose strain for high-level protein expression and easy induction. The BL21(DE3)pLysS competent cells provide tighter control of protein expression for expression of toxic proteins and are resistant to chloramphenicol.

Can BL21 (DE3) cells be used to express non-toxic heterologous genes?

For best results, use BL21(DE3) competent cells to express non-toxic heterologous genes. Because of the extremely high activity ofT7 RNA polymerase, some basal level expression of the gene ofinterest may occur in uninduced cells. This creates problems incases where the gene of interest is toxic to bacterial cells.

What is the function of the DE3 lysogen and the plasmid plyss?

BL21 (DE3)pLysS E. coli carry both the DE3 lysogen and the plasmid pLysS. pLysS constitutively expresses low levels of T7 lysozyme, which reduces basal expression of recombinant genes by inhibiting basal levels of T7 RNA polymerase. For Research Use Only. Not for use in diagnostic procedures. Convenient, on-site access to the products you need.