Part:BBa_K2952014

Formate dehydrogenase (FDH_h) facilitates this reaction: CO2 + NADH <-> HCOO- + NAD+. Formic acid is a chemical commodity and can be easily stored and transported, making it a stable form of hydrogen fuel. It can be converted into hydrogen gas by E.coli's native formate hydrogenlyase. This formate dehydrogenase has high binding affinities for NADH and CO2, and is expected to generate formate efficiently.

Our modelling suggested that overexpression of FDH_h should increase hydrogen production more than any other hydrogenase.

Figure 1: Hydrogen (H2) flux for various E. coli strains growing on different media with a single carbon source using flux balance analysis (FBA). Biomass growth rate was set as the objective, while the uptake rate for carbon sources was set to -20mmol/gDWh. This shows that FDH_h-expressing E. coli out-performs all other strains, while galactose was the best carbon source.

The transcriptional unit was assembled using loop assembly. Successful assembly was confirmed using diagnostic digestion and sequencing.

FDH_h was over-expressed in E. coli BL21DE3 using T7 promoter (BBa_I712074). Successful expression after induction with IPTG was confirmed by his-tag purification and subsequent SDS-PAGE.

Figure 2: FDH_h was purified using Ni-affinity resin. Supernatant (S/N), flow trough 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. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8µM.

Activity was assayed through NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH_h can be expressed in E. coli while still maintaining functionality.

Figure 3: NADH oxidation assay was carried out using purified FDH protein. 0.1 M sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls (0.1mg/ml of protein was added to the reaction mixture described above). This assay showed that recombinant FDH_h can be expressed in E.coli and retain functionality.