Extended Data Fig. 2: A Cre recombinase-based indicator of transposition frequency at the population level.

Following outgrowth of attTn7 site-specific integrants, an optional second conjugation step can be performed to determine the population-level frequency of mini-Tn5 transposition out of the attTn7 site. When a plasmid expressing the Cre recombinase is introduced into the mutant population, Cre expression leads to excision of the Tn5 transposition complex at the attTn7 site via recombination of the lox sequences. Cre excision causes recipient cells to simultaneously lose the attTn7-site kanamycin marker and activate expression of the gentamicin marker. Cells that did not undergo mini-Tn5 transposition prior to Cre excision of the Tn5 transposition complex become solely resistant to gentamicin, while cells that did undergo transposition retain a copy of the mini-Tn5 transposon at a random genomic ___location outside of the mini-Tn7 lox sites, rendering them resistant to both kanamycin and gentamicin. The ratio of gentR+kanR to gentR colonies provides a measure of transposition frequency in cells where the Tn5 transposition complex was initially integrated at the attTn7 site (Fig. 2b).