Escherichia coli BL21(DE3)
| Escherichia coli BL21(DE3) | |
|---|---|
Scientific classification
| |
| Domain: | Bacteria |
| Phylum: | Pseudomonadota |
| Class: | Gammaproteobacteria |
| Order: | Enterobacterales |
| Family: | Enterobacteriaceae |
| 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. This strain combines several features that allow for excessive expression of heterologous proteins. It is derived from the B lineage of E. coli.[1]
Naming
[edit]The genotype of this strain is designated with E. coli B F– ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS).[2]
Characteristics
[edit]Decreased proteolysis
[edit]The proteolysis of heterologously expressed proteins is reduced due to the functional deficiency of two major proteases, Lon and OmpT.[3] 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.[4]
Expression induction
[edit]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.[5]
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.[6] 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.[7]
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).[8]
Facilitated cloning
[edit]E. coli BL21(DE3) lacks a functional type I restriction-modification system, indicated by hsdS(rB- mB-). 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.[9]
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'.[10] This facilitates further processing of purified DNA as Dcm methylation prevents cleavage by certain restriction enzymes.[11]
References
[edit]- ^ a b "What are E. coli Expression Strains?". Goldbio. Retrieved 2024-06-07.
- ^ "E. coli genotypes". OpenWetWare. Retrieved 2024-06-07.
- ^ "BL21(DE3)". EcoliWiki. Retrieved 2024-06-06.
- ^ "CGSC Strain#: 12504". Coli Genetic Stock Center. Retrieved 2024-06-07.
- ^ "How Does IPTG Induction Work?". Goldbio. Retrieved 2024-06-07.
- ^ "T7 RNA Polymerase". Promega. Retrieved 2024-06-07.
- ^ "BCH-GENE-SCBD-258850-1". Biosafety Clearing-House. Retrieved 2024-06-07.
- ^ "Genetic backgrounds of each Escherichia coli strain used in The ST2OOL Project" (PDF). IGEM. Retrieved 2024-06-07.
- ^ "EcoKI restriction-modification system". EcoCyc. Retrieved 2024-06-07.
- ^ "P0AED9 · DCM_ECOLI". UniProt. Retrieved 2024-06-07.
- ^ "Dam and Dcm Methylases of E. coli". New England Biolabs. Retrieved 2024-06-07.