Fig. 3
Targeted insertion and expression of the T. pseudethanolicus 39E adhE (Teth39_0206) and adhB (Teth39_0218) in C. bescii. a A diagram of the integration vector pDCW180 (Additional file 1: Figure S1), which contains the PS-layerTeth39_0206 expression cassette and pyrF cassette [27] for selection of transformants. Homologous recombination can occur at the upstream- or downstream-targeted chromosomal regions (inter-cistronic region between Cbes0863 and Cbes0864), integrating the plasmid into the genome and generating uracil prototroph strain. Counter selection with 5-FOA selects for loss of the plasmid sequences but not the adhE expression cassette. Bent arrows depict primers used for verification of the integrated expression cassette. Apr apramycin resistance gene cassette. b Gel depicting PCR products amplified from the targeted chromosome region in JWCB001 (wild-type; lane 1), JWCB017 (ΔpyrFA Δldh; lane 2), JWCB049 (ΔpyrFA Δldh CIS1:: PS-layerTeth39_0206; lane 3), and JWCB054 (ΔpyrFA Δldh CIS1:: PS-layerTeth39_0218; lane 4) amplified by primers DC477 and DC478. M1: 1-kb DNA ladder (New England Biolabs). c Total cell protein lysates (75 µg) isolated from mid-log phase cultures grown at 75 °C were electrophoresed in SDS-PAGE gels, followed by Western blot analysis probed with anti-His-tag antibody as described in “Methods”. Lane 1: JWCB017; lane 2: JWCB049; lane 3: JWCB054; M2: MagicMark™ XP Western Protein Standard (Invitrogen)