Losito, OF; (2013) Doing without antibiotics: the pMB1 origin of replication as a novel selectable marker in enteric bacteria. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Plasmids are used in the biopharmaceutical industry for the production of recombinant proteins, recombinant DNA for DNA vaccines and as non-viral vectors for gene therapy. Antibiotic- resistance genes are used for plasmid selection and to ensure plasmid maintenance. Regulatory bodies like the European Medicines Agency have introduced guidelines to limit the use of antibiotics and their resistance genes in the manufacture of therapeutics. Therefore, it is important to develop novel, effective methods for antibiotic-free plasmid selection. Cobra Biologics has designed a novel mechanism for antibiotic-free plasmid selection called oriSELECT. oriSELECT uses RNAI and RNAII, the antisense RNAs of the pMB1 plasmid origin of replication (ori) as the selectable marker. These two RNAs are involved in replication and its control of almost all plasmid vectors used commercially and academically. By placing the RNAII sequence in the E. coli chromosome upstream of either a toxic gene or a repressor for an essential gene, cell growth should be inhibited. In cells containing a plasmid with the pMB1 replicon producing RNAI, transcription of the growth inhibitor is blocked allowing cell growth and survival. This work describes the testing of various cassettes useful for oriSELECT and the construction of oriSELECT strains.

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