The ORT® technology employs a plasmid-mediated repressor titration to activate a host selectable marker, removing the requirement for a plasmid-borne marker gene. Current ORT® strains are engineered to contain an essential gene, dapD under transcriptional control of the lac operator/promoter (lacO/P), although any essential gene could be used. In the absence of an inducer such as IPTG, this strain cannot grow due to the repression of dapD by the LacI repressor protein binding to lacO/P. Transformation with a high copy number plasmid containing the lac operator (lacO) effectively induces dapD expression by titrating LacI from the operator. Regulation of the essential gene ensures the growth of bacteria and maintenance of recombinant plasmids containing lacO and an origin of replication.
Figure 1: An ORT® cell without a plasmid will lyse due to the lack of dapD expression. When transformed with a multi-copy plasmid, this titrates the repressor, enabling dapD expression and therefore plasmid selection and maintenance.
In addition to plasmid DNA production, the ORT® system has been adapted for vaccine delivery using live bacterial vectors as ORT-VAC by Prokarium Ltd.
ORT® is used along with X-mark™, which enables a plasmid to be constructed using highly efficient antibiotic selection in a special E. coli strain, then transformed into an ORT® strain, which rapidly excises the antibiotic resistance gene by Xer recombination to generate a 1kb smaller plasmid which is stably maintained by ORT®. This greatly accelerates the time for generating plasmids in ORT® strains.
Figure 2: The antibiotic resistance gene in a plasmid is flanked by psi sites and their accessory sequences, which are recognised by the native Xer recombinases. The plasmid is structurally stable as it is constructed using a pepA E. coli mutant which cannot perform Xer recombination on plasmids. It is then transformed into an ORT strain under antibiotic selection. When cultured without the antibiotic, the antibiotic resistance gene is excised to generate a selectable marker gene-free plasmid.