Xer-cise for Bacteria
Plasmids are usually the best vehicles for gene expression in E. coli due to their elevated copy number, but in some species, such as Bacillus subtilis, single-copy chromosomal genes provide sufficient levels of secreted protein production. Both plasmid transformation and chromosomal integration usually require the use of resistance markers.
To circumvent the use of antibiotics and marker genes, constructs have traditionally been engineered to first allow selection for the gene insertion and then, in a second step, to remove the marker using site-specific recombinases. Typically these constructs use recombinase systems such as Cre/loxP derived from bacteriophage P1 or Flp/FRT from yeast. However, these systems display varied success rates with much efficiency lost through the need to express exogenous recombinases in a range of bacterial species.
Cobra Biomanufacturing (www.cobrabio.com) has developed a new excision system, Xer-cise, which provides a rapid method for inserting genes into or deleting genes from bacterial chromosomes. The Xer-cise system uses Xer recombinases, naturally present in a wide range of bacteria, to excise antibiotic-resistance genes. This eliminates the need to express exogenous site-specific recombinases.
In a proof-of-concept case study the gene for bovine pancreatic RNase (rbpA) was introduced into a plasmid containing the antibiotic resistance gene cat flanked by Xer recombination sites. After chromosomal insertion of the rbpA-cat cassette through homologous recombination, the positive transformants were selected by antibiotic resistance. Removal of the antibiotic from the growth medium resulted in the elimination of the marker gene by the endogenously expressed Xer recombinase (Figure 1). In contrast to traditional systems, Xer recombinase is endogenously expressed in a majority of prokaryotes, making it ideal for the engineering of difficult strains, or strains that have a limited molecular biology toolkit available.