Team:Edinburgh UG/Results

Hydrogen Collection Results

Figure X: Graph of hydrogen produced in moles per hour for Rhodobacter sphaeroides WT (triangle) and Rhodobacter sphaeroides HPCA (circle) as grown in 250 mL cultures at 30oC in m22 media under continuous bright light.

Formate dehydrogenase (FDH)

We assembled and characterised formate dehydrogenase from Clostridium carboxidivorans. Loop assembly was used to construct the transcriptional unit and transformed into E. coli strain BL21DE3. Successful assembly and transformation was checked through diagnostic digestion and sequencing (figure 1).

Figure 1: Assembled plasmid was digested with SacII, expected bands were 1.8kb and 3kb

Figure 2: Supernatant (S/N), flow through 1 and 2 (FT1/FT2), and eluted fractions 1 and 2 were run on a 10% SDS-PAGE gel. The expected size of our protein is 80.7kDa.

His-tagged FDH protein expression in E. coli was confirmed through immobilised metal affinity chromatography (IMAC) and subsequent SDS-PAGE analysis. Bands were visualised after destaining and were in the expected region of 80.7 kDa. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8 µM.

Activity was assayed via NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH can be expressed in E. coli while still maintaining functionality. The assay was carried out using purified FDH protein. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls.

Figure 3: 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. 0.5mg/ml of protein from soluble fractions of controls were added to the reaction mixture described above. All measurements were done in triplicates and averaged.