Expression systems are important tools in molecular biology, enabling researchers to produce proteins for research, diagnostics, and therapeutics. Examples of E. coli protein expression strains are BL21 cells, BL21 (DE3) cells and DL39(DE3) cells. But what exactly does that DE3 mean?
The “DE3” in BL21(DE3) and DL39(DE3) means that these strains contain the lambda DE3 prophage that has the gene for T7 RNA polymerase which is under the control of the lacUV5 promoter. This allows high-level expression of genes downstream of a T7 promoter through IPTG induction.
That answer could sound a bit technical, so in this article, we’re breaking it all down for you including how to choose which strain is right for you.
Article Table of Contents
Why is DE3 important for recombinant protein expression?
What’s the difference between BL21(DE3) and DL39(DE3)?
Are there any drawbacks to using DE3-based strains?
Cell stress from overexpression
When should you choose a DE3 strain for protein expression?
Choosing between BL21 and BL21 (DE3) Cells
BL21 vs. BL21 (DE3) summary table
Quick strain comparison [BL21, BL21 (DE3), and DL39 (DE3)]
How DE3 relates to T7 RNA polymerase and the λ prophage?
What is the DE3 prophage?
The DE3 prophage is a piece of DNA that scientists have inserted into the chromosome of certain E. coli strains like BL21 and DL39. This DNA comes from a modified virus called lambda (λ) phage, but it's been engineered so it can’t harm the bacteria or make more viruses.
The neat thing about this inserted DNA is that it has a gene that makes T7 RNA polymerase – an enzyme that reads DNA and transcribes it into RNA. But this gene doesn’t turn on by itself. It’s placed under the control of the lacUV5 promoter, which only turns on when ITPG is added to your cells (inducible expression via IPTG).
This setup lets scientists control exactly when bacteria start making a protein, and it helps them make a lot of it too.
Ultimately, this means that the DE3 strains are a popular choice for protein production workflows.
Why is DE3 important for recombinant protein expression?
The main advantage of DE3 competent cell strains is that they allow the expression of genes under the T7 promoter—which gives researchers tight control over protein expression using the LacUV5 promoter and IPTG induction.
T7 RNA polymerase transcribes DNA much more efficiently than native E. coli RNA polymerase, leading to higher yields of recombinant protein.
What’s the difference between BL21(DE3) and DL39(DE3)?
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BL21(DE3) is the most widely used DE3 strain and is derived from the E. coli B strain. It lacks lon and ompT proteases, which helps reduce recombinant protein degradation.
DL39(DE3) is a lesser-known but related derivative that may carry additional mutations beneficial for specific expression challenges, such as tighter regulation or reduced background expression. Always look at the literature to confirm specific differences and use cases.
We actually dive in a lot deeper about the differences between these two strains along with the genetic features of expression strains like these cells in this article.
Are there any drawbacks to using DE3-based strains?
There are some drawbacks to using DE3-based competent cell strains. While these cells have some great expression features, they also have limitations like leaky expression, and burden from high levels of expression.
Leaky expression
Leaky expression can be an issue. Even without IPTG induction, small amounts of T7 RNA polymerase can sometimes be made, which means your target gene may be expressed too early. This can be especially problematic if your protein is toxic to E. coli — it could slow down cell growth or even kill the cells before you have a chance to induce and collect your protein. However, leaky expression can be limited in DE3-based strains by using a defined growth medium (Studier, 2005).
Cell stress from overexpression
Another drawback is cell stress from overexpression. The T7 system is so strong that when it’s turned on, the cell puts a lot of energy into making your target protein. While this sounds great, in reality it can overwhelm the cell’s folding and quality control systems. The result is that your protein may end up in inclusion bodies—clumps of misfolded protein that are inactive and hard to recover.
There’s also the issue of metabolic burden. Overexpressing a foreign protein uses a lot of the cell's resources—ATP, amino acids, chaperones, etc.—which can slow down cell growth or change cell behavior.
This makes timing and optimization of IPTG really important. Often, by reducing the amount of IPTG used for protein induction you can avoid the overexpressed protein going into inclusion bodies and causing metabolic burden.
So, while DE3-based strains are excellent for high-level expression, they require careful handling and optimization to avoid issues that can impact yield or protein quality.
When should you choose a DE3 strain for protein expression?
Use a DE3 strain when your plasmid has a T7 promoter, and you need high levels of protein expression. DE3 strains are especially useful for large-scale protein production, structural biology studies, or enzyme activity assays. If your protein is toxic or expressed at low levels, you can try optimizing lower IPTG induction, but ultimately you might need to use strains with tighter controls or co-expression chaperones.
How DE3 relates to T7 RNA polymerase and the λ prophage?
T7 RNA polymerase is a phage-derived enzyme known for its high specificity and transcriptional efficiency. In DE3 strains, the gene encoding T7 RNA carries only the genes needed to express T7 RNA polymerase but not the genes needed to undergo lytic replication.
So, another way to explain this is that where the phage would normally take over the cell’s machinery to make more viruses and eventually lyse the cell, the λDE3 prophage in DE3 strains is modified so it can’t do this. Lacking these features helps supercharge protein production.
The lacUV5 promoter regulates T7 RNA polymerase expression. When IPTG is added to the culture, it induces expression of T7 RNA polymerase, which then recognizes and transcribes genes cloned under the control of a T7 promoter on a compatible plasmid.
This two-tiered control system enables robust expression of target genes while minimizing leaky expression in uninduced cultures.
The role of IPTG induction
IPTG (isopropyl β-D-1-thiogalactopyranoside) is a lactose analog that induces the lac operon but is not metabolized by E. coli. When added to cultures of BL21(DE3) cells or DL39 (DE3) cells, IPTG binds to the lac repressor, lifting repression from the lacUV5 promoter.
This triggers T7 RNA polymerase gene expression, which in turn initiates transcription of the target gene on the plasmid. Because this cascade is tightly regulated, researchers can grow cultures to high densities before induction, maximizing protein yield while minimizing cellular stress.
Choosing between BL21 and BL21 (DE3) Cells
Choosing between BL21 and BL21(DE3) depends on a few factors, like the type of plasmid you’re using, the protein you’re working with, and how you want to express your protein.
Use BL21 If:
- Your plasmid does not use a T7 promoter.
- You’re using a different expression system (like a lac, pBAD, or trc promoter).
- You need very low background expression of your gene (for example, if your protein is toxic).
- You want a general-purpose strain for cloning, small-scale expression, or baseline comparisons.
BL21 is good for basic experiments or expressing less toxic proteins at lower levels.
Use BL21 (DE3) If:
- Your plasmid uses a T7 promoter (e.g., from the pET vector family).
- You want high-level expression of a recombinant protein.
- You’re ready to induce expression with IPTG.
- You’re optimizing production for large-scale purification or functional studies.
BL21 vs. BL21 (DE3) summary table
Table 1. Situational lookup table for BL21 and BL21 (DE3) cells
|
Situation |
Best Choice |
Why |
|
Non-T7 promoter plasmid |
BL21 |
No need for T7 polymerase |
|
T7 promoter plasmid |
BL21(DE3) |
Required for expression |
|
Toxic protein, need tight control |
BL21 (or DL39(DE3)) |
Lower risk of leaky expression |
|
High-yield protein expression |
BL21(DE3) |
Strong, IPTG-inducible T7 system |
|
Basic cloning or maintenance of plasmids |
BL21 |
Less metabolic burden on cells |
Quick strain comparison [BL21, BL21 (DE3), and DL39 (DE3)]
Table 2. Compares the three strains: BL21, BL21(DE3), and DL39(DE3)
|
Feature |
BL21 |
BL21(DE3) |
DL39(DE3) |
|
T7 RNA polymerase gene |
No |
Yes (inserted via λDE3 prophage) |
Yes (inserted via λDE3 prophage) |
|
Inducible protein expression |
No (not T7-compatible) |
Yes, IPTG-inducible T7 expression |
Yes, IPTG-inducible T7 expression |
|
Recommended for T7 plasmids |
No |
Yes |
Yes |
|
Protease deficiency |
Lacks lon and ompT proteases |
Same as BL21 |
Check supplier specs |
|
Background expression |
Low |
Moderate (some leaky T7 expression) |
Lower than BL21(DE3) (engineered for tighter control) |
|
Use case |
General cloning, low-background |
High-yield expression with T7 system |
Optimized T7 expression with tighter regulation |
|
Expression control |
No T7 control |
IPTG control via lacUV5 promoter |
IPTG control, with improved regulation (strain-specific) |
Note: DL39(DE3) is often used in research settings that need tighter expression control or different expression characteristics than BL21(DE3).
Ultimately, when it comes to the “DE3” designation in BL21(DE3) and DL39(DE3) strains, it signifies the presence of a prophage that carries the gene for T7 RNA polymerase under IPTG-inducible control. This setup is really helpful for expressing proteins. Understanding how DE3 functions allows researchers to optimize expression workflows and avoid common pitfalls in protein production.
For IPTG, DE3 strains, and more resources to support your protein expression work, browse GoldBio for high-quality competent cells, reagents and technical support.
