Extended Data Fig. 4: Mating-out assay to monitor transposition of ISGst3.
From: Transposon-encoded nucleases use guide RNAs to promote their selfish spread
a, Schematic of mating-out assay, in which transposition events into the F-plasmid are monitored via drug selection. E. coli donor cells carrying an F-plasmid were transformed with a plasmid encoding either TnpA and ISGst3-derived mini-Tn (pDonor1) or an autonomous ISGst3 element with tnpA and tnpB (pDonor2). After induction of TnpA, conjugation was used to transfer the F-plasmid into the recipient strain, and transposition events were quantified by selecting for recipient cells (RifR) containing spectinomycin (F+) and kanamycin (mini-Tn+) resistance. b, Transposition frequency of ISGst3 mini-Tn deriving from pDonor1 into the F-plasmid was measured with and without tnpA. Bars indicate mean ± s.d. (n = 6). c, Drug-selected cells from mating-out assays from pDonor1 contain TAM-proximal IS insertions, as evidenced by long-read Nanopore sequencing. A genetic map of the F-plasmid is shown, along with the location of distinct ISGst3-derived mini-Tn integration events. The insets show a zoom-in view of each integration site at the nucleotide level, with the TAM highlighted in yellow and the integration site denoted by an arrow. d, Transposition frequency of an autonomous ISGst3 deriving from pDonor2 into the F-plasmid was measured with active and catalytically inactive forms of TnpA and TnpB. M, TnpA Y125A mutant; d, TnpB D196A mutant; ND, not detected. Bars indicate mean ± s.d. (n = 3).
