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.

Naming
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).

Decreased proteolysis
The proteolysis of heterologously expressed proteins is reduced due to the functional deficiency of two major proteases, Lon and OmpT. 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.

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.

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. 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.

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.

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).

Facilitated cloning
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.

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'. This facilitates further processing of purified DNA as Dcm methylation prevents cleavage by certain restriction enzymes.