Figure 5.

TnsA, TnsB, TnsC, TniQ and a new open reading frame TnsF are required for comM att targeting in the heterologous E. coli host. (A) Schematic representing the general steps of the mating-out assay protocol. A mini-Tn6022 element harboring kanamycin resistance is integrated into the attTn7 site on the E. coli chromosome in donor cells (yellow) which also harbor a conjugal plasmid with a 600 bp portion of comM on it. Following a period of induction to produce transposition proteins, donor cells are mated with a population of counter-selectable recipient cells (red). Recipient cells are plated on selective media to calculate the number of exconjugants that received a conjugal plasmid with an integrated mini-element vs. total exconjugants to determine transposition frequency. (B, C) Percent transposition as measured by the mate-out assay using different combinations of Tn6022 transposition proteins in the presence of a conjugal plasmid harboring a 600 bp region of comM (‘comM’). (D) Sanger sequencing demonstrates precise ‘comM’ targeting which exactly recapitulates insertions found in nature. A DNA chromatogram demonstrating Sanger sequencing of Tn6022 insertions within ‘comM’ obtained from recipient cell colonies that were mated with donors expressing TnsABC + TniQ + TnsF. 10 isolates were screened for insertions via PCR using a pair of ‘comM’ flanking primers before sequencing. In all 10 isolates the transposon ends are clearly distinguished interrupting comM along with the hallmark target-site duplication (TSD) which occurs as a result of transposition. All experiments were performed with 0.02% arabinose induction to express the full length TniQ + TnsF operon, and 0.1mM IPTG to express the full length TnsABC operon. TnsE experiments were performed with 0.0002% arabinose to express TnsE. All experiments were performed in triplicate with error bars representing the standard error of the mean. Created with BioRender.com.