Figure 2

Deletion of the ldh gene in C. bescii . (A) A deletion cassette for the ldh gene was constructed in a non-replicating plasmid that contained a wild type copy of the pyrF gene, resulting in plasmid pDCW121. The cassette contained ldh 5′ and 3’ flanking DNA fragments. The plasmid was transformed into JWCB005, and uracil prototrophs were selected (resulting from plasmid insertion). Counter-selection with 5-FOA selected for strains that underwent a second recombination event, resulting in deletion of the marker and ldh to produce strain JWCB017. (B) Agarose gel showing PCR products amplified from the ldh locus in the wild type (lane 2), JWCB005 (ΔpyrFA parent strain, lane 3) and JWCB017 (ΔpyrFA Δldh, lane 4). Lane 1: 1 kb DNA ladder; Lane 5: no template PCR control. Expected bands: wild type ldh locus – 3 kb; ldh deletion – 2.0 kb.