E. coli expression strains, BL21(DE3) and DL39 for example, are used for recombinant RNA/protein expression. They have the necessary genetic features such as lacking proteolytic activity and having a T7 RNA polymerase gene controlled by the lacUV5 promoter.
In this article, we will focus on BL21(DE3) and DL39 as important examples of E. coli expression strains and will explore the necessary genetic features in expression strains that make them appropriate for mass-producing recombinant therapeutic products.
Article table of contents:
Key genetic features of the BL21(DE3) expression strain
Genetic feature 1: T7 RNA polymerase and the T7 expression system
Genetic feature 2: Lacking Lon and OmpT proteases
BL21(DE3) transformation efficiency and genetic manipulation
Limitations using BL21(DE3) as an expression strain
The auxotrophic background of the DL39 E. coli expression strains
Key genetic features of the BL21(DE3) expression strain
The BL21(DE3) expression strain is one of the most widely used E. coli strains for protein expression. It has two primary genetic features, among others, necessary for mass expressing the desired end-product of the cloned recombinant DNA.
We’ll break down both of those features below.
Genetic feature 1: T7 RNA polymerase and the T7 expression system
The first genetic feature of BL21(DE3) is a special RNA polymerase with a very high transcription rate, so the cloned DNA is transcribed in high quantities.
This special RNA polymerase is called the T7 RNA polymerase, which is under the control of a promoter called the lacUV5 promoter.
Figure 1. Schematic showing the T7 RNA polymerase gene arrangement in the E. coli genome.
More specifically, the BL21 E. coli strain is derived from E. coli B strains, which are known for their favorable characteristics in genetic studies. BL21(DE3) is a specific derivative that carries the DE3 lysogen.
The DE3 lysogen is a prophage (lysogen) containing the T7 RNA polymerase gene under the transcriptional control of the lacUV5 promoter.
This prophage has been acquired in this strain through a process called lysogeny, where the lambda prophage containing the T7 RNA polymerase gene downstream of the lacUV5 promoter has integrated into the E. coli chromosome.
Transcription from the lacUV5 promoter is activated by IPTG (isopropyl β-D-1 thiogalactopyranoside). So, when BL21 (DE3) is grown in the absence of IPTG, the T7 RNA polymerase is usually repressed. And in turn, no significant amounts of the target protein are produced.
However, when IPTG is added to the culture medium, it acts as an inducer that causes the T7 RNA polymerase to be expressed from the host strain’s genome.
As a result, high levels of target proteins are produced downstream of the T7 promoter (a promoter from which transcription is activated by T7 RNA Polymerase) in the plasmid expression vector.
Figure 2. Represents an E. coli BL21(DE3) expression strain with the DE3 lysogen (orange) in the E. coli genome (purple).
To sum this part up, DE3 refers to a lambda prophage that encodes the T7 RNA polymerase gene. This gene and its promoter are integrated into the host E. coli strain’s genome.
BL21(DE3) strains, when transformed with a plasmid carrying the target gene downstream of the T7 promoter, produce T7 RNA polymerase from their genome upon IPTG induction.
The T7 RNA polymerase then recognizes the T7 promoter and drives the transcription of the target gene, resulting in high-level expression of the recombinant RNA or protein.
The plasmid with a T7 promoter is called an expression vector, which is different from a cloning vector used to clone the recombinant DNA in a previous step.
We have a detailed article about cloning vectors and E. coli cloning strains that can help you further understand the differences between these two types of strains.
So, taken together, the T7 expression system comprises the:
- T7 RNA Polymerase downstream of the IPTG inducible lacUV5 promoter in the expression strain’s genome
- Expression vector that has the transgene cloned in it downstream of a T7 promoter.
Now we will look into the pivotal features of why this setup is central to the BL21(DE3) expression strain’s functionality in terms of high-level transcription of the cloned transgene.
High-level expression:
The T7 promoter is strong and tightly regulated. When transcription is activated from this promoter by T7 RNA Polymerase, the target gene is transcribed with a high expression level – about 5 to 10 times that of the transcription rate of E. coli’s own RNA Polymerase (Springman, Badgett, Molineux, & Bull, 2005).
This leads to the production of a large amount of the cloned recombinant RNA or protein, making it valuable for applications requiring high yields of the target recombinant product.
Inducible expression: The T7 promoter is indirectly tightly controlled by the lacUV5 promoter, which responds to the presence of the inducer IPTG. IPTG is a lactose analog that binds to the lac repressor and releases its repression on the lacUV5 promoter, leading to the T7 RNA polymerase expression.
In turn, this IPTG-induced T7 RNA polymerase drives transcription of the cloned transgene from the T7 promoter in the plasmid (figure 3). This allows the researcher to control the timing and amount of recombinant RNA/protein production by regulating the concentration of IPTG in the growth medium.
Figure 3. Schematic of T7 RNA Polymerase system. 1. T7 RNA polymerase downstream of a PlacUV5promoter in the E. coli genome can be activated by adding IPTG to the culture. 2. This leads to the synthesis of the T7 RNA Polymerase enzyme. 3. This enzyme drives transcription of the gene of interest (gene) cloned downstream of a T7 RNA polymerase-responsive promoter in the expression vector.
Specificity:
The T7 RNA polymerase is highly specific to the T7 promoter and does not recognize E. coli’s endogenous promoters effectively.
This means that when the T7 RNA polymerase is expressed in BL21(DE3) cells, it selectively transcribes the target gene under the control of the T7 promoter, minimizing unintended transcriptional interference with other cellular processes.
Versatility:
The T7 promoter system is versatile because it allows researchers to use various T7 expression vectors carrying different target genes.
You can easily clone your target gene downstream of the T7 promoter in compatible expression vectors and transform them into BL21(DE3) cells for efficient protein expression.
Genetic feature 2: Lacking Lon and OmpT proteases
The second feature is that BL21 lacks some proteases (Lon and OmpT) that would otherwise break down the recombinant protein.
Lon protease is a cytoplasmic protease that degrades foreign proteins while OmpT is an outer membrane protease that degrades extracellular proteins.
Expression strains lacking these proteolytic genes ensures that the recombinant products are not degraded after synthesis and can be harvested.
BL21(DE3) transformation efficiency and genetic manipulation
BL21(DE3) strains have a high transformation efficiency, which is beneficial when introducing plasmids carrying your gene of interest. This facilitates the efficient incorporation of recombinant DNA constructs into the host cells.
Additionally, BL21(DE3) strains are amenable to various genetic manipulations, allowing researchers to modify the strain for specific needs, such as introducing specific mutations or incorporating additional expression elements.
BL21(DE3) growth characteristics favorable for synthesis and harvesting recombinant RNA and protein products
Not only do the genetic factors, ease of use, and high transformation efficiency make BL21(DE3) cells ideal expression strains, but there are also key features that aid in important elements of protein production.
BL21(DE3) is a well-characterized and optimized strain, allowing for rapid growth in standard bacterial growth media. It has a relatively short doubling time, typically around 20-30 minutes, which means it can quickly reach high cell densities during the exponential growth phase.
The rapid growth characteristic makes it suitable for producing moderate to high yields of protein enabling their purification and subsequent downstream applications such as structural studies, biochemical analyses and initial protein characterization.
Another important feature is that BL21(DE3) strains are widely used to express soluble proteins.
Finally, they are compatible with various expression vectors, such as pET vectors, which provide additional elements for protein expression, purification and detection.
Limitations using BL21(DE3) as an expression strain
Although BL21(DE3) has many advantages, there are some limitations to consider.
First, the strain lacks certain proteases such as Lon and OmpT. While this is advantageous during recombinant protein expression, this can lead to an accumulation of unstable proteins. Fortunately, this limitation can be addressed by using specific variants or modified strains.
Additionally, BL21(DE3) is mainly used for cytoplasmic protein expression, and the production of membrane proteins or proteins requiring extensive post-translational modifications may require alternative expression systems.
In summary, the BL21(DE3) expression strain is a widely utilized E. coli strain for protein expression. Its strong points include
- high transformation efficiency,
- rapid growth,
- the ability to perform inducible protein expression using the T7 system.
By employing the BL21(DE3) strain, researchers can efficiently produce large quantities of recombinant proteins.
DL39 Expression strain
DL39 is an engineered E. coli expression strain that has attracted attention for its improved capabilities in recombinant RNA/ protein production. This strain has a genetic modification akin to the other expression strain BL21(DE3) strain-–the T7 expression system. The other feature capitalized extensively in this strain is its auxotrophic mutations for some amino acids. This becomes very handy in expressing some specific recombinant proteins.
Here is a detailed overview of the DL39 expression strain, highlighting its features helpful in recombinant protein expression.
The auxotrophic backgroundof the DL39 E. coli expression strains
DL39 is an auxotrophic mutant for amino acids phenylalanine, tyrosine, aspartic acid, leucine, isoleucine, and valine. This feature can be helpful both during gene cloning- by providing selectable markers. More importantly, these mutations are utilized when mass producing certain types of recombinant proteins.
For example, scientists might want to produce a recombinant protein tagged with fluorescent amino acids of a specific type such as tyrosine, as in this study (Olson et al. 2023)
Here, DL39 would be extremely helpful for this purpose, because it cannot synthesize tyrosine on its own; and depends on the supply of this amino acid in the culture medium.
Fluorinated tyrosine, instead of regular non-fluorinated tyrosine would be supplied in the growth media for this recombinant protein’s expression. The DL39 expression strain would grow using the exogeneous fluorinated tyrosine supplemented in the media – as it cannot synthesize tyrosine on its own, because of the auxotrophic mutation. These fluorinated tyrosine residues would be incorporated in the desired recombinant protein that this strain mass produces.
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DL39 growth characteristics
DL39 maintains rapid growth characteristics typical of E. coli strains. This fast growth rate is advantageous for efficient propagation of transformed cells and large-scale production of recombinant proteins.
The strain is amenable to standard culture conditions and media used for E. coli, making it readily accessible to researchers in the biotechnology community.
Transformation efficiency
DL39 retains a high transformation efficiency, enabling the efficient integration of plasmids carrying the desired target gene. The ability to easily introduce recombinant DNA constructs into host cells is crucial for successful protein expression experiments.
The high transformation efficiency of DL39 streamlines the cloning process and contributes to its widespread use in various laboratories.
Protein expression
DL39 has demonstrated impressive protein expression capabilities, making it a preferred choice for numerous research projects. Like BL21(DE3), this strain has the genetics of T7 expression system (figure3).
In summary, DL39 is a valuable addition to the repertoire of E. coli expression strains. It offers:
- improved growth characteristics,
- high transformation efficiency,
- auxotrophic mutations for amino acids phenylalanine, tyrosine, aspartic acid, leucine, isoleucine, and valine
Researchers working in the field of recombinant protein production can benefit from DL39’s advanced features because it allows for reliable and efficient generation of valuable proteins for various biotechnological applications, including therapeutic protein production, industrial enzyme production, and structural biology studies.
As the field of biotechnology continues to evolve, both BL21(DE3) and DL39’s contributions to protein expression are expected to play a pivotal role in advancing scientific discoveries and biotechnological innovations.
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