RecTE_Psy-mediated recombination frequency is sensitive to flank length.

<p>(A) Recombination substrates were generated by PCR using primers that flank the ΔPSPTO_1203::<i>neo</i> allele to produce substrates with homologies of indicated lengths. For example, each of the substrates is composed of the <i>neo</i> gene flanked by the indicated amount of genomic sequence. The substrate was then gel purified and 0.46 pmol of each substrate was used to electroporate cells containing the RecTE_Psy expression vector or empty vector (0.46 pmol corresponds to 200 ng of the 80 bp flanks substrate). (B) Recombination frequencies with these substrates in wild-type and <i>recA</i>⁻<i>P. syringae</i> pv. <i>tomato</i> DC3000 with pUCP24/recTE are shown. The results are the average of at least three independent replicates, error bars indicate standard deviation. No kanamycin resistant colonies were observed in control transformations of cells containing the pUCP24/61 empty vector (no RecTE_Psy). PCR was used to confirm that for each length tested the kanamycin resistance gene had integrated into the correct location.