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Antibiotic-free Maintenance System (ORT®)

Cobra's Antibiotic-free Maintenance System can provide potential benefits for gene therapy and DNA vaccine products.

Advantages of ORT®

Gene Therapy - A plasmid backbone with an improved safety profile that maximises the efficiency of therapeutic gene delivery of therapeutic genes

DNA Vaccines - Smaller plasmids for improved antigen gene delivery

ORT® can offer numerous advantages when compared with the use of antibiotics and selectable marker genes.

  • 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 associated with selectable marker gene expression

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)

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)

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.

References & Publications

Cranenburgh RM 2013. Operator-Repressor Titration: Stable Plasmid Maintenance without Selectable Marker Genes. In Minicircle and Miniplasmid DNA Vectors. Wiley-Blackwell. p. 7-21.

Durany O, Bassett P, Weiss AME, Cranenburgh RM, Ferrer P, Lopez-Santin J, de Mas C and Hanak JAJ, 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 RM, Lewis KS and Hanak JAJ, 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 RM, Hanak JAJ, Williams SG, and Sherratt DJ, 2001. Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration. Nucleic Acids Res. 29: e26.

Hanak JAJ and Cranenburgh RM, 2001. Antibiotic free plasmid selection and maintenance in bacteria. In Recombinant protein production with prokaryotic and eukaryotic cells. Kluwer Academic Publishers. p. 111-124.

Williams SG, Cranenburgh RM, Weiss AME, Wrighton CJ, Sherratt DJ and Hanak JAJ, 1998. Repressor titration: a novel system for selection and maintenance of recombinant plasmids. Nucleic Acids Res. 26: 2120-2124.

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