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Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria

Salerno, P; Leckenby, MW; Humphrey, B; Cranenburgh, RM

Authors

P Salerno

MW Leckenby

B Humphrey

RM Cranenburgh



Abstract

Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark (TM), a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins.

Citation

Salerno, P., Leckenby, M., Humphrey, B., & Cranenburgh, R. (2022). Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria. Synthetic Biology, 7(1), https://doi.org/10.1093/synbio/ysac005

Journal Article Type Article
Acceptance Date Apr 12, 2022
Publication Date May 13, 2022
Deposit Date Aug 23, 2022
Publicly Available Date Aug 23, 2022
Publisher Oxford University Press
Peer Reviewed Peer Reviewed
Volume 7
Issue 1
DOI https://doi.org/10.1093/synbio/ysac005
Keywords plasmid; antibiotic resistance; E. coli; Salmonella; X-mark; SITE-SPECIFIC RECOMBINATION; ESCHERICHIA-COLI; AMINOPEPTIDASE-A; COPY NUMBER; SELECTION; SYSTEM; TRANSFORMATION; INSTABILITY; ENCODES; DNA

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