Figure 1
Targeted insertion and expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene in C. bescii. a A diagram of the integration vector pDCW171 (see Additional file 1: Figure S1 for details), which contains the PS-layerTeth39_1957 expression cassette and pyrF cassette for selection of transformants. Homologous recombination can occur at the upstream or downstream targeted chromosomal regions, integrating the plasmid into the genome and generating a strain that is a uracil prototroph. Counter-selection with 5-FOA selects for loss of the plasmid sequences, but not the Teth39_1957 expression cassette. Bent arrows depict primers used for verification of the integrated expression cassette. Ap r apramycin resistance gene cassette. b Gel depicting PCR products amplified from the targeted chromosome region in JWCB001 (wild-type; lane 1), JWCB018 (ΔpyrFA ldh::ISCbe4 ΔcbeI; lane 2), JWCB044 (ΔpyrFA ldh::ISCbe4 ΔcbeI :: P S-layer Teth39_1597; lane 3), and no DNA (lane 4) amplified by primers DC477 and DC478. M: GeneRuler 1 kb DNA ladder (Thermo Scientific). c, d Total cell protein lysate (80 µg) isolated from mid-log phase cultures were electrophoresed in SDS-PAGE gels either for staining with Coomassie Brilliant Blue (c) or for Western blot analysis (d) probed with His-tag antibody as described in “Methods”. Lane 1 JWCB001 grown at 75°C, lane 2 JWCB018 grown at 75°C, lane 3 JWCB044 grown at 65°C, lane 4 JWCB044 grown at 70°C, lane 5 JWCB044 grown at 75°C, M1 Pre-stained SDS-PAGE standards, Broad range (Bio-Rad Laboratories) (c), M2 MagicMark™ XP Western Protein Standard (Invitrogen) (d).