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. 2016 Aug 22;44(18):8921–8932. doi: 10.1093/nar/gkw724

Figure 3.

Figure 3.

Bxb1 Int recombination of two supercoiled plasmids. Integrase reactions using supercoiled substrates with attP and attB produce recombinant products with multiple knot nodes. Int reactions were performed at 37°C with ∼50 nM pAttP and pAttB supercoiled plasmids in standard reaction buffer containing spermidine (10 mM) to increase reaction efficiency and product yield. After the times indicated, samples were treated with the nicking enzyme Nb.BsrNDI (New England Biolabs) at 65°C for 60 min and then with SDS and proteinase K. Products were analyzed by electrophoresis (45 V, 20 h) on a 1% agarose-E buffer gel. The gel was subsequently stained with ethidium bromide to visualize the DNA. (A) Reaction contained pAttP.TA and pAttB.TA, both 2.73 kb. (B) Reactions contained pAttP.GC (3.1 kb) and pAttB.GC (3.5 kb) (lanes 2–4, counting from left), pAttP.CT (3.1 kb) and pAttB.CT (3.5 kb) (lanes 5–6), or pAttB.GT (3.5 kb) and pAttP.GT (3.1 kb) (lanes 7–8), and were run for the times indicated. (C) A reaction of pAttP.CT and pAttB.CT in which both parents were pre-nicked to remove supercoiling before Int addition. M indicates 1 kb ladder. The unknotted recombinants, the recombinant torus knots (31, 51, 71, 91, 111, …) and the 2-noded catenane of the parental plasmids are shown. Note that the 6.2 kb dimeric form of the 3.1 kb pAttP plasmids migrates at the same position as the 2-noded catenane.