Escherichia coli BL21(DE3)

Escherichia coli BL21(DE3)
Scientific classification
Domain:	Bacteria
Kingdom:	Pseudomonadati
Phylum:	Pseudomonadota
Class:	Gammaproteobacteria
Order:	Enterobacterales
Family:	Enterobacteriaceae
Tribe:	Escherichieae
Genus:	Escherichia
Species:	E. coli
Strain:	E. c.  BL21(DE3)
Trionomial name
Escherichia coli BL21(DE3)

Escherichia coli BL21(DE3) is a commonly used protein production strain of the E. coli bacterium. This strain combines several features that allow for excessive expression of heterologous proteins. It is derived from the B lineage of E. coli. ^[1] More specifically, it is derived from E. coli BL21 by the addition of a lambda prophage element called DE3. ^[2]

Naming

The genotype of this strain is designated with E. coli B F^– ompT gal dcm lon hsdS_B(r_B^–m_B^–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB⁺]_K-12(λ^S). ^[3]

Characteristics

Decreased proteolysis

The proteolysis of heterologously expressed proteins is reduced due to the functional deficiency of two major proteases, Lon and OmpT. ^[4] Lon is usually present in the cytoplasm of the cell, but in all B strains its production is prevented by an insertion within the promoter sequence. OmpT is located in the outer membrane but is absent in B strains due to deletion. ^[5]

Expression induction

While E. coli BL21(DE3) supports the expression of genes under the control of constitutive promoters, it is specifically engineered for IPTG induction of recombinant genes under the control of a T7 promoter. The realized induction strength depends on several factors, including the IPTG concentration and the timing of its supplementation. ^[6]

This function is enabled by the presence of a recombinant λ-prophage (DE3). DE3 carries a T7 RNA polymerase (RNAP) gene under the control of a lacUV5 promoter (lacUV5-T7 gene 1). T7-RNAP is highly specific to the T7 promoter and orthogonal to native E. coli promoters. Therefore the T7-RNAP only transcribes (exogenously introduced) genes that are regulated by a T7 promoter. ^[7] The LacUV5 promoter is derived from the E. coli wildtype lac promoter but exhibits an increased transcription strength due to two mutations that facilitate its interaction with a native E. coli RNAP σ-factor. ^[8]

In E. coli BL21(DE3) the expression of the T7-RNAP is suppressed by the constitutively expressed LacI repressor. LacI binds the lac operator, which is located downstream of the LacUV5 promoter, preventing the production of the T7-RNAP. However, upon supplementation of IPTG, the LacI repressor dissociates from the lac operator, allowing for the expression of T7-RNAP. Subsequently, T7-RNAP can initiate the transcription of a recombinant gene under T7 promoter control. ^[1]

Other DE3 modifications ensure stable integration of the prophage in the genome and prevent the prophage from entering the lytic cycle (ind1, sam7, and nin5). ^[9]

Facilitated cloning

E. coli BL21(DE3) lacks a functional type I restriction-modification system, indicated by hsdS(r_B⁻ m_B⁻). Specifically, both the restriction (hsdR) and modification (hsdM) domains are inactive. This enhances transformation efficiency since exogenously introduced unmethylated DNA remains untargeted by the restriction-modification system. ^[10]

The dcm gene is also rendered inactive, preventing the methylation of a cytosine on both strands within the recognition sequence 5'-CC(A/T)GG-3'. ^[11] This facilitates further processing of purified DNA as Dcm methylation prevents cleavage by certain restriction enzymes. ^[12]

Related strains

As mentioned earlier, this strain is directly descended from BL21. B-derived strains in general have the decreased proteolysis and facilitated cloning benefits as described before. As a result, a lot of work has poured into developing them for protein production, and BL21(DE3) is far from the only one. ^[2]

• BL21, aforementioned.
• BL21-AI, Invitrogen's strain that uses a T7 expression system driven by the tightly regulated arabinose-inducible P_BAD promoter. It fixes the leaky expression issue seen in BL21 and BL21(DE3). ^[13]
• BL21Star(DE3), a derivative of BL21(DE3) with a truncation in the rne gene (RNase), which leads to more protein produced. ^[14]
• C41/43(DE3), C44/45(DE3) and Mt56(DE3), three attempts at evolving a version of BL21(DE3) more resistant to product toxicity. They have reduced production rates. ^[14]
• Evo21(DE3), a mutant of BL21(DE3) produced by "constrained evolution", featuring a truncation in rre. Produces more protein and is more resistant to toxicity. ^[14]

References

1. "What are E. coli Expression Strains?". Goldbio. Retrieved 2024-06-07.
2. Jeong, Haeyoung; Kim, Hyun Ju; Lee, Sang Jun (30 April 2015). "Complete Genome Sequence of Escherichia coli Strain BL21". Genome Announcements. 3 (2). doi:10.1128/genomea.00134-15.
3. "E. coli genotypes". OpenWetWare. Retrieved 2024-06-07.
4. "BL21(DE3)". EcoliWiki. Retrieved 2024-06-06.
5. "CGSC Strain#: 12504". Coli Genetic Stock Center. Retrieved 2024-06-07.
6. "How Does IPTG Induction Work?". Goldbio. Retrieved 2024-06-07.
7. "T7 RNA Polymerase". Promega. Retrieved 2024-06-07.
8. "BCH-GENE-SCBD-258850-1". Biosafety Clearing-House. Retrieved 2024-06-07.
9. "Genetic backgrounds of each Escherichia coli strain used in The ST²OOL Project" (PDF). IGEM. Retrieved 2024-06-07.
10. "EcoKI restriction-modification system". EcoCyc. Retrieved 2024-06-07.
11. "P0AED9 · DCM_ECOLI". UniProt. Retrieved 2024-06-07.
12. "Dam and Dcm Methylases of E. coli". New England Biolabs. Retrieved 2024-06-07.
13. Bhawsinghka, N; Glenn, KF; Schaaper, RM (5 March 2020). "Complete Genome Sequence of Escherichia coli BL21-AI". Microbiology resource announcements. 9 (10). doi:10.1128/MRA.00009-20. PMID   32139577.
14. Heyde, Sophia A. H.; Nørholm, Morten H. H.; Haberland, G (12 August 2021). "Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity". Communications Biology. 4 (1). doi:10.1038/s42003-021-02493-4.