cobra:bio

• Native Xer recombinases excise the antibiotic resistance gene after chromosomal insertion
• No exogenous recombinases (e.g. Cre, Flp) are required
• Xer-cise works in a broad range of bacterial species
• Xer-cise enables multiple gene integration events in the same strain

• Gene deletion: precise excision of target genes prevents reversion in mutant strains
• Gene insertion: new genes can be inserted to alter the phenotype, or for protein expression

The deletion and insertion of genes in bacterial chromosomes has traditionally been accomplished by labelling the integration cassette with an antibiotic resistance gene, thus selecting the mutants by antibiotic resistance. The disadvantages of this approach are:

• Permanent insertion of a foreign gene can alter the expression of surrounding genes
• If an antibiotic resistance gene is present on the chromosome, the same gene cannot be used for plasmid maintenance
• Multiple gene integration events become difficult due to the limited number of suitable antibiotic resistance genes

To address this, antibiotic resistance genes have been flanked with the sites for site-specific recombinase enzymes, which have to be supplied in trans on a plasmid. This requires an additional transformation step, followed by further culturing to remove the helper plasmid. Additionally, this approach has only been optimised for a limited range of bacteria.

The Xer-cise technology employs native Xer recombinases that normally function to restore the chromosomal and plasmidal dimers generated by RecA back to monomers. These enzymes are present in the vast majority of bacterial species. An antibiotic resistance gene is flanked by the dif sites, which are in turn flanked by chromosomal target homology. This cassette is either constructed on a plasmid and linearized, or assembled by PCR, and transformed into the target bacterium. Gene integration mutants are selected on agar plates containing the antibiotic. These are then cultured in antibiotic-free medium, and the Xer recombinases recombine the two dif sites to a single site, thereby excising the intervening antibiotic resistance gene to generate the new mutant strain (Figure 1). Xer-cise has been used successfully for gene insertions and deletions in E. coli, Salmonella and Bacillus subtilis, and is currently being evaluated in a range of other species. Cobra offers the technology for licensing, or fee-for-service bacterial genetic modification.





Figure 1. Chromosomal gene integration and antibiotic resistance gene (cat) excision by Xer-cise.

Bloor, A. E. and Cranenburgh, R. M., 2006. An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl. Environ. Microbiol. 72: 2520-2525.