Antibiotic-free Maintenance System (ORT®)

ORT® (Operator-Repressor Titration): A method of producing
plasmid DNA without antibiotics, antibiotic resistance genes
or any other selectable marker genes in Escherichia coli.

Features and benefits

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

Applications

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

Background

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

Technical design

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).

References

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. 


Over 320 batches for Phase I, II, III clinical trials and commercial supply


 

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