Figure 5. The ability of different rsaA constructs to inhibit supercoil diffusion does not correlate with their ability to establish a chromosomal domain boundary.

1. A γδ TnpR recombinase (green) was induced from a cumate‐inducible promoter. TnpR recombines two res sites (blue and red arrows) that flank a tet resistance cassette (orange arrows) to excise the intervening DNA. Recombination requires supercoiling‐dependent juxtaposition of res sites.
2. Induction of TnpR from P_cumate results in a loss of cell viability on tetracycline plates. Each spot is a 10‐fold dilution.
3. Percentage of tetracycline‐resistant colonies represent a proxy for the extent of supercoil diffusion inhibition by the various P_rsaA‐rsaA constructs indicated, each inserted between one res site and the tet ^R cassette. Error bars represent SD, n = 3. The ability of each construct to form a CID boundary is indicated on the left (see Figs 2 and 3).
4. LacI binding to 10 consecutive lacO sites in between two res sites does not significantly inhibit supercoil diffusion. TnpR and LacI‐YFP (brown arrow) were induced from P_cumate and P_xyl, respectively. Error bars represent SD, n = 3.