ORT® provides antibiotic-free maintenance of high copy number plasmids
ORT® plasmids are highly stable
No potential for residual antibiotic contamination of plasmid DNA
Minimise the size of the backbone plasmid DNA to allow more therapeutic gene per milligram of DNA
No metabolic burden due to selectable marker gene expression
DNA vaccines: Smaller plasmids for improved antigen gene delivery
Gene therapy: A plasmid backbone with an improved safety profile that maximises the relative delivery of therapeutic genes
Recombinant protein production: Antibiotic-free fermentation with potential for enhanced product yields through enhanced stability
The isolation of bacterial transformants and subsequent stable plasmid maintenance has traditionally been accomplished using plasmid-borne selectable marker genes. The marker genes typically encode resistance to an antibiotic such as ampicillin, or alternatively encode an essential gene that complements a host cell deficiency (auxotrophy). The disadvantages of these systems are:
Requirement of plasmid-borne gene transcription
Imposition of a metabolic burden on the host
Potential reduction in the yield of the recombinant protein particularly with high copy number plasmids
Use of antibiotics in large scale fermentation leading to additional cost and downstream processing requirements to remove traces of antibiotics
Durany, O., Bassett, P., Weiss, A. M. E., Cranenburgh, R. M., Ferrer, P., Lopez-Santin, J., de Mas, C. and Hanak J. A. J., 2005. Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations. Biotechnol. Bioeng. 91: 460-467.
Cranenburgh, R. M., Lewis, K. S. and Hanak, J. A. J., 2004. The effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by Operator-Repressor Titration in Escherichia coli. J. Mol. Microbiol. Biotechnol. 7: 197-203.
Cranenburgh, R. M., Hanak, J. A. J., Williams, S. G., and Sherratt, D. J., 2001. Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration. Nucleic Acids Res. 29: e26
In addition to plasmid DNA production, the ORT®system has been adapted for vaccine delivery using live bacterial vectors.