Deletion of the gene encoding putative restriction enzyme ChyI. A) Scheme for targeted gene deletion. The pJGW03 chyI knockout vector is transformed into JWCH006 (ΔpyrF), and uracil prototrophy selects for integration at one of the 500-bp flanking regions, denoted by the gray boxes. 5' integration is shown. The uracil prototroph is then plated on 5-FOA to select for loop-out of the pyrF cassette via homologous recombination between flanking region sequences. The two possible results are a return to the wild-type sequence or a clean chyI deletion. (B) Chromosomal map of the locus containing the ORF for chyI. The deleted region is indicated by the line below the diagram. Bent arrows depict primers used to verify deletion of chyI in the JWCH008 strain. (C) Gel depicting PCR products of the chyI region in the JWCH006 ΔpyrF strain (2.3 kb) compared to the JWCH008 ΔpyrF ΔchyI strain (1.25 kb) amplified by the indicated primers (JG025 and JG028). 1: C. hydrothermalis JWCH006 genomic DNA; 2: C. hydrothermalis JWCH008 genomic DNA; 3: negative control; M: 1 kb DNA ladder (NEB).