Expression of foreign proteins in microbial cells is crucial for molecular microbiology. One of the best-known and highest-performing systems is pET/BL21; this consists of a pET plasmid, containing the T7 promoter, and the E. coli BL21 (DE3) host strain, containing the T7 polymerase; these work together to enable both high expression levels and tight control of expression.

Despite their wide usefulness, the pET plasmids are not perfect; they contain much junk DNA, they do not have modular construction, are incompatible with modern cloning methods, have some “leaky” expression, and are sold under restrictive licences. Here we worked on designing improved and open-source versions of the pET plasmid. These consisted of replication, resistance, repressor, and cloning site modules made from synthetic DNA to enable removal of junk DNA, internal restriction sites, and cryptic promoters.

The initial synthetic pET plasmid constructed (pMQ1A) gave higher IPTG-inducible GFP expression than pET28. However, pMQ1A showed very leaky expression in the absence of inducer. This problem was addressed in a second version (pMQ1B) by increasing the expression of the lac repressor gene. Future work will investigate the effect of lower or controllable replication origins on plasmid stability and expression levels.