Increasing the deletion size has little effect but increasing the substrate size indicates limits to beta recombination. (A) Schematic illustration of an assay using a BAC carrying variously sized insertions in the neo gene, from 4 to 4000 bps as indicated, which were deleted by a 100 bp fragment to restore kanamycin resistance. Results are plotted at the right. (B) Schematic illustration of an assay using a BAC assay with one homology arm (red) placed at various distances from 0.5 to 50 kb to the other homology arm (red) as indicated. The region between these two sequences was deleted by insertion of a 500 bp fragment carrying blasticidin resistance as plotted to the right. (C) Schematic illustration of an assay to insert either 1, 2 or 3 kb fragments carrying the neo gene into the same place on a BAC. The fragments were synthesized to have 5' phosphorylated (P) or phosphothioated (S) ends as indicated in the plotted curves to the right. (D) Schematic illustration of an assay showing the insertion of a 1kb fragment carrying the rpsL and blasticidin (bsd) genes into the same site on a BAC. The 1kb fragment was identical except that the homology arms were arranged for ends-out (replacement, above) or ends-in (below) recombination, and synthesized with combinations of 5' phoshorylated (P) or phosphothioated (S) ends as indicated in the plot to the right. The experiments were performed in our standard host (GB2005 = WT) or the same host after deletion of ruvabc.
Maresca et al. BMC Molecular Biology 2010 11:54 doi:10.1186/1471-2199-11-54