X-mark™ – rapid, automatic deletion of antibiotic resistance genes from plasmids
Antibiotic resistance genes are very useful for plasmid construction, but are not permitted for use with live bacterial vectors for reasons of biosafety. Even if they were allowed, their constitutive expression causes a metabolic burden to the bacteria that can lead to plasmid loss.
Prokarium’s X-mark™ technology works in a plasmid in which the antibiotic resistance gene is flanked by psi sites and their accessory sequences, which are recognised by the native Xer recombinases and are required for recombination on plasmids but not on chromosomes. This means that an E. coli mutant that cannot produce one of the accessory proteins, PepA, can still resolve chromosome dimers (so remains viable) but cannot carry out Xer recombination on plasmids. The plasmid is constructed using a pepA E. coli mutant and is transformed into an ORT-VAC strain of Salmonella (see below) under antibiotic selection. When cultured in the absence of antibiotic selection, the antibiotic resistance gene is excised to generate a selectable marker gene-free plasmid.