ORT-VAC™ – selectable marker-free plasmid maintenance

Prokarium’s ORT-VAC™ technology (Operator-Repressor Titration for Vaccines) is the only selectable marker-free plasmid stabilisation technology that can function with any type of plasmid, enabling recombinant antigens to be delivered orally. With ORT-VAC Salmonella, an essential gene on the bacterial chromosome is engineered such that its expression is controlled by a repressor protein, which is also encoded on the chromosome. Under normal growth conditions in culture and in the gastrointestinal tract, the essential gene is maintained in a de-repressed state by the binding (titration) of any free cytoplasmic repressor by the short non-coding operator sequence carried on the plasmid. Therefore in the absence of the plasmid the ORT-VAC strain cannot grow, as reduced plasmid copy number leads to increased levels of cytoplasmic repressor and shut down of the essential gene function. Plasmids are thus maintained without the use of any selective marker gene. A useful by-product of the approach is that neither the plasmids nor the host can survive in isolation and therefore cannot disseminate in the environment; another pre-requisite for a recombinant, live bacterial product.