Selection of hyperactived Bin and Tn21 catalytic domains.

<p><b>(A)</b> Schematic representation of the split gene reassembly system used to evaluate recombinase activity. Expression of an active recombinase leads to excision of the stuffer fragment (GFPuv), restoration of the β-lactamase reading frame and host cell resistance to carbenicillin (right, bottom). Full-length ZFR target site is shown and consists of a 20-bp core sequence recognized by the recombinase catalytic domain flanked by zinc-finger binding sites. Core positions are numbered. <b>(B, C)</b> Recombination activity of the native Bin, Tn21, and hyperactivated β, Gin, Sin and Tn3 catalytic domains in the context of ZFRs. Activity was measured on 20-bp core sites flanked by zinc-finger binding sites. Each core site was derived from the native <b>(B)</b> Bin and <b>(C)</b> Tn21 recombination sites (referred to as 20-Bin and 20-Tn21, respectively). Recombination was determined by split gene reassmbley as the percentage of recombined carbenicillin and chloremphenicol resistant clones versus total chloremphenicol resistant clones. Error bars indicate standard deviation (<i>n</i> = 3). <b>(D, E)</b> Selection of <b>(D)</b> Bin and <b>(E)</b> Tn21 variants that recombine 20-bp core sites derived from their native recombination sites, 20-Bin and 20-Tn21, respectively. Each recombinase catalytic domain was randomly mutated by error-prone PCR and analyzed for activity as a ZFR on a 20-bp core site-flanked by zinc-finger binding-sites. Asterisks indicate selection steps in which incubation time was deceased from 16 to 4 hr.