"(A) A diagram of the pyrBCF locus in the C. <t>bescii</t> chromosome. The line above the diagram depicts the extent of the spontaneous deletion in the Δ pyrBCF strain. pDCW70 contains the <t>wild</t> <t>type</t> pyrBCF alleles with an engineered KpnI site used to select marker replacement of the deletion depicted with primers used to confirm the structure of the chromosome in the transformant. B) Δ pyrBCF electrocompetent cells after electro-pulse with no DNA added plated onto defined medium + uracil (top plate), Δ pyrBCF electrocompetent cells after electro pulse with unmethylated pDCW70 DNA plated onto defined medium w/o uracil (middle plate), Δ pyrBCF electrocompetent cells after electro-pulse with M.CbeI methylated pDCW70 DNA plated onto defined medium w/o uracil (bottom plate). (C) PCR products amplified using primers DC163 and DC188 (upper gel) digested with KpnI (lower gel). M: 1kb DNA Ladder (NEB). lane 1: amplified from wild type cells (3.2 kb); lane 2: amplified from Δ pyrBCF (1.63 kb); lane 3: amplified from transformants (1.9 and 1.3 kb cleavage products). "