What are BL21 Competent Cells?

by Simon Currie, Ph.D.

BL21 competent cells are an E. coli strain that is specifically engineered to excel at recombinant protein expression. There are different types of BL21 to choose between depending on the expression vector and transformation strategy that you’re using.

Bacteria are a cornerstone of modern molecular biology. For example, over 70% of the approximately 240,000 protein structures that have been solved to date have come from recombinant proteins expressed in Escherichia coli (Protein Data Bank, 2025). Which means that before these proteins from humans and other organisms were purified to study their structure, they were expressed in E. coli.

In addition to protein expression, E. coli are also very useful in molecular cloning. However, there are different E. coli strains that are particularly well suited for these distinct purposes. That’s why it’s important to know which kind of E. coli strain you should work with for your intended application.

If you’ve been browsing different E. coli strains you’ve probably seen BL21 cells show up in your search. This article will be a great resource if you’ve been wondering what these cells are and if they’re appropriate for your experimental needs.

BL21 competent cells are an E. coli strain that is specifically engineered to excel at recombinant protein expression. There are different types of BL21 to choose between depending on the expression vector and transformation strategy that you’re using.

Why are BL21 cells good for protein expression?

Why are BL21 such a good E. coli strain for protein expression?

BL21 cells have been engineered to delete the genes that encode for the proteases Lon and OmpT. These proteases degrade proteins, including your protein of interest that you’re trying to overexpress.

By eliminating these proteases from the cells, you can generate larger quantities of your protein (Figure 1).

illustration of e. coli cells vs. bl21 competent cells

Figure 1. Regular E. coli (left) has proteases (pink) which degrade your protein of interest (orange) while it’s being expressed. BL21 (right) lack Lon and OmpT proteases which leads to more abundant protein production.

As an analogy, this would be like if you were writing thank you cards next to someone with scissors. If by the time you had written five cards they had cut up three of them, your progress would be quite slow. Removing the scissor-wielder from this process is essentially like having BL21 cells with proteases deleted.

Adding protease inhibitors to your buffers during cell lysis and your initial purification step helps prevent protein degradation, and is a good idea even when using BL21 cells. However, protein expression in BL21 helps prevent protein cleavage even before that point in the purification, while the protein is being expressed in live cells since the Lon and OmpT proteases are absent.

BL21 vs BL21(DE3)

Ok, so now that you know that BL21 cells are great for expressing protein, you still need to decide what kind of BL21 cells will be best for your specific purpose. This means deciding between regular BL21 cells or BL21(DE3) cells.

The take-home point here is that if you are using a plasmid with a T7 promoter, then you will want to use BL21(DE3) cells. This is because DE3 stands for the lambda DE3 prophage. This is a piece of DNA from the lambda phage that contains a gene encoding for T7 RNA polymerase and is stably integrated into the BL21 genome. Then, you add IPTG to your bacterial culture which will trigger the expression of the T7 RNA polymerase, leading to transcription of your gene of interest (Figure 2).

Figure 2. IPTG induction stimulates transcription of T7 polymerase by relieving inhibition on the lac promoter (left). T7 polymerase then transcribes plasmids with a T7 promoter to generate the protein of interest (right). Double arrows indicate intermediate RNA transcription and translation steps between gene and protein.

pET vectors are frequently used plasmids with a T7 promoter, so if you are using a pET vector, or any other kind of plasmid with a T7 promoter then BL21(DE3) cells are the ones you should use (Heyde & Nørholm, 2021).

Alternatively, if you’re using a plasmid with a promoter other than T7, then there is no reason to use BL21(DE3) and you should just use regular BL21 cells instead.

We’re just skimming the surface here about how this process works. So, if you want more details about protein expression check out these articles for more details about promoters, DE3 strains, IPTG induction, and protein expression.

Electrocompetent vs chemically competent cells

Ok, now that you’ve figured out whether to use BL21(DE3) or regular BL21 cells, the next important choice is how you’re going to transform your cells.

In this context, transformation refers to delivering your gene of interest into the bacterial cells.

There are two main options for transforming bacterial cells: electroporation and chemical transformation.

Both methods are pretty simple and quick. Transformation efficiencies tend to be higher for electroporation compared to chemical transformation (Aune & Aachmann, 2010). However, transformation efficiency is almost never a limiting factor for protein expression studies, so either method typically works well when transforming BL21 cells.

Electroporation does require a specialized instrument to deliver the electric shock, whereas chemical transformation only requires a heated water bath, something that most labs already have.

For either transformation method, you first incubate the DNA with your gene(s) of interest with the BL21 cells on ice for about 30 minutes. This gives time for the DNA to localize to the outside of the BL21 cells.

During electroporation, you give the bacteria a quick zap of electricity, which permeates the membranes and allows the DNA to transit inside the cells. Then you add E. coli competent cell recovery medium to the BL21 cells and warm them up to 37°C to help them recover from their shock.

Chemically competent cells have been treated with chemicals, such as calcium chloride, to make their membranes more permeable to DNA. For chemically, competent cells you use heat shock to transform the cells by incubating them in a 42°C water bath for 40 seconds. During this heat shock, the DNA with your gene of interest will enter the bacterial cells. Then, you put your tubes back on ice and recover with E. coli competent cell recovery medium as described above.

For more details about cell transformation and the difference between electrocompetent and chemically competent cells, check out our Introduction to Competent Cells and guide to Understanding Competent Cells for Bacterial Transformation.

Overall, BL21 cells are a great choice for producing recombinant proteins. GoldBio has a few different BL21 cell strains, which are listed in the related section below to find which ones you need: BL21 or BL21(DE3), electrocompetent or chemically competent.

Additionally, we have a lot of related articles about cell transformation, DE3 strains, IPTG induction, and more, so check those out below as well. May all your transformations be true, and all of your proteins express great!

Related Resources

Introduction to Competent Cells

What Does the DE3 Represent in Competent Cells Like BL21(DE3) and DL39(DE3)

Understanding Competent Cells for Bacterial Transformation

How Does IPTG Induction Work?

5 Key Features of Plasmids Explained

References

Aune, T. E. V., & Aachmann, F. L. (2009). Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed. Applied Microbiology and Biotechnology, 85(5), 1301–1313. https://doi.org/10.1007/s00253-009-2349-1.

Heyde, S. A. H., & Nørholm, M. H. H. (2021). Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity. Communications biology, 4(1), 963. https://doi.org/10.1038/s42003-021-02493-4

Protein Data Bank. (2025, August 26). PDB Statistics: PDB Data Distribution by Expression System Organism. https://www.rcsb.org/stats/distribution-expression-organism-gene