Deletion of the celA gene in Caldicellulosiruptor bescii . (A) Depiction of the chromosomal location of celA and construction of the gene deletion. A deletion cassette was constructed in a non-replicating plasmid, pJFW52, which contained a wild-type copy of the pyrF gene for prototropic selection of transformants in a strain containing a pyrFA deletion. The cassette contained celA 5' and 3' flanking DNA fragments. The plasmid was transformed into JWCB018 (ΔpyrFA ΔcbeI), and uracil prototrophs were selected (resulting from plasmid insertion). Counter-selection with 5-FOA selected for strains that underwent a second recombination event, some of which resulted in deletion of celA to produce strain JWCB029 (ΔpyrFA ΔcbeI ΔcelA). (B) Agarose gel showing PCR products amplified using primers JF200 and DC432 from the celA locus in the parent strain JWCB018 (lane 2) and the the celA deletion strain, JWCB029 (lane 3). Lane 1: DNA MW standards; lane 4: no template PCR control. Expected bands: wild-type celA locus: 7.1 kb; celA deletion: 1.8 kb. (C) SDS-PAGE gel stained with Coomassie Brilliant Blue showing precipitated extracellular protein from the wild-type JWCB001 (lane 3), JWCB018 (lane 5), and JWCB029 (lane 7), standards (Precision Plus Protein™ Dual Color Standards; BioRad) in lane 1.