Expression of foreign proteins in microbial cells is crucial for many biotechnology applications. One of the best-known and highest-performing systems used for protein expression is pET/BL21; this consists of the pET plasmid and the E.coli BL21(DE3) strain, which use a combination of the T7 phage promoter and the lac repressor to enable both high expression levels and tight control of expression.
The pET plasmid has several design flaws and other practical problems; it contains much junk DNA, it does not have modular construction, it is incompatible with modern cloning methods, it has some 'leaky' expression in the uninduced state, and it is sold under a restrictive licence. Here we used a synthetic biology approach to rebuild the pET plasmid to resolve these problems and limitations.
The pET28 backbone was recreated from synthetic DNA, in the process removing junk DNA, restriction sites, and cryptic promoters. Each module (replication origin, resistance gene, cloning site, lac repressor) was provided with independent promoters and terminators, and conserved adapters for PCR priming or Gibson assembly were added between modules. The Standard European Vector Architecture (SEVA) design was adopted for maximum flexibility of use. The full T7 promoter was restored (this is truncated in most pET plasmids), and a stronger terminator was added downstream of the cloning site. A free-use GFP gene (fuGFP) was cloned into the plasmid to test its functionality.
The replication and resistance modules of the synthetic pET plasmid (here named pMQ1A) functioned as expected, and the new vector gave higher levels of induced expression compared to the original pET28. However, the uninduced levels of expression were much higher than pET28, possibly due to the stronger variant of the T7 promoter used in pMQ1A. This problem was solved by enhancing the strength of the promoter driving the lac repressor gene to yield plasmid pMQ1B. We expect this plasmid to be broadly useful in molecular microbiology and biotechnology research.