Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement.