Team:Edinburgh UG/Experiments

Formate dehydrogenase (FDH)

For our new biobrick contribution, we decided to assemble and characterise formate dehydrogenase (FDH). This enzyme facilitates this reversible reaction between carbon dioxide and formate: 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 hydrogen lyase [footnote 1]. This formate dehydrogenase has high binding affinities for NADH and CO₂, and is expected to generate formate efficiently [footnote 2].

The transcriptional unit was assembled using loop assembly. Successful assembly was confirmed using diagnostic digestion and sequencing. His-tagged FDH was over-expressed in E.coli strain BL21DE3 using a T7 promoter. Successful expression after induction with IPTG was confirmed by Ni-affinity chromatography column and subsequent SDS-PAGE analysis.

Activity was assayed through NADH oxidation at 37° and was carried out using purified FDH protein. 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. This showed that recombinant FDH can be expressed in E.coli while still maintaining functionality.

Brewing co-product - spent grain

We also had the pleasure to receive distillery by-product. While this made the lab smell a bit like a brewery (and our supervisor wasn't too pleased either...) we were able to prepare the co-product to culture both strains of Rhodobacter with the intention of also testing this carbon source with our modified organism. As reducing costs of our technology is a main aim of our project, we wanted to extend this theme to all aspects of our project’s design. When speaking with Scottish Gas Network, the ability to use co-product sources in remote areas was flagged as a potential cost cutting solution in certain rural areas. Find out more about our integrated Human Practices

Hydrogen Collection

Our initial setup relied on capturing all gas produced by simple water displacement. This provided a proof of concept and a rough benchmark for how much gas we could expect to produce, but since our culture produced both carbon dioxide and hydrogen this couldn’t give us a quantitative measurement of hydrogen.

Throughout our project we've tried to maintain a theme of frugality as we want to be able to produce a cost effective source of green energy. We thought using a high cost analytical technique such as gas chromatography would go against this; especially since in order to do this it would be necessary to use headspace analysis which would be even more costly. We wanted a cost effective analytical method so we headed out of the lab and back to the drawing board to improve our method.

The New & Improved

Combining a 2007 Paper by C Zanchetta and colleagues as well as work from the 2017 Macquarie iGEM team allowed for an efficient and cost effective method that produced a quantitative measurement while aligning with our theme of frugality and our human practices [1,2]. This allowed us to use barium hydroxide as Macquarie did to remove carbon dioxide while also being able to carry out hydrogen collection in a closed system.

When dissolved in water carbon dioxide creates carbonic acid. Carbonic acid reacts rapidly with barium hydroxide forming an extremely insoluble white precipitate; barium carbonate. By passing the gas produced into a flask containing barium hydroxide (0.1 M) it was possible to precipitate carbon dioxide at the interface as barium carbonate.

Equation 1: Reaction of barium hydroxide and carbon dioxide1

Since the carbon dioxide had been stripped from the gaseous phase the only other gas produced would be hydrogen. The production of hydrogen would lead to an increase in pressure inside the flasks which would, in turn, lead to a force being exerted on the liquid forcing it through the tubing. This results in the water level in the syringe rising in line with the Bernoulli principle. Since under standard conditions an ideal gas takes up 22.4 L this change in water level could then be converted back into moles of hydrogen using the ideal gas law.

Kieran preparing solution in a fume hood for our new collection methods.

Equation 1: Ideal gas law rearranged to give number of moles hydrogen as a function of pressure volume and temperature [1].

However, this equation comes from a system running a catalysed reaction resulting in a far greater change in pressure than will be seen on our scale. As such, it is possible to state that P0 ≈ P(t), this greatly simplifies the equation.

Equation 2: Simplified ideal gas law equation for number of moles hydrogen.

The pressure of the system will be equal to atmospheric pressure minus that of the vapour pressure of water at 30 degrees C. This method thereby allowed for cost efficient analysis while also being perfect for the sensitive small scale experiment necessary.

In order to monitor gas production as closely as possible AutoHotKey programming was used to create a script that would take pictures of the syringe at set time intervals [3].

Promoter Characterisation

We decided to characterise PA1/04/03 lac based synthetic promoter by assembling it with strong R. sphaeroides RBS (BBa_J95018), GFP (BBa_E0040) and terminator (BBa_B1006) using loop assembly. The promoter and RBS were shown to work in R. sphaeroides with high expression rates (Huo, 2011). We tested if this promoter-RBS combination could yield high expression rates in E. coli as this would provide a convenient system for parallel expression in both R. sphaeroides and E. coli. Three 5ml liquid cultures containing recombinant E.coli DH5α having R. sphaeroides RBS (BBa_J95018), GFP (BBa_E0040), terminator (BBa_B1006) and Kanamycin resistance gene were incubated in a 37°C shaker overnight. Next day, these cultures were diluted to 0.05 OD600 and incubated at 37° to the limit where they had 0.5 OD600. Total of 100μl culture was induced with 8 different IPTG concentrations- (5μM, 10μM, 20μM, 30μM, 45μM, 60μM, 120μM and 180μM) and measured at an excitation and emission of 485nm and 520nm. Orbital averaging was set to 3 and the gain to 1300. Cell growth was measured at 600nm. The fluorescence was divided by the approximated number of cells to get normalised fluorescence per cell.

Figure 1. GFP fluorescence per cell

References

C Zanchetta, B Patton, G Guella, and A Miotello, 2007, An integrated apparatus for prodyction and measurement of molecular hydrogen, IOP Publishing
Macquarie iGEM Team, 2017, HydroGEM
https://github.com/A-Sobieska/Edinburgh_UG_iGEM_2019
Huo, Junling, "Design of a BioBrickTM Compatible Gene Expression System for Rhodobacter sphaeroides" (2011). All Graduate Theses and Dissertations. 877. https://digitalcommons.usu.edu/etd/877

Footnotes

Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. Enhanced Hydrogen Production from Formic Acid by Formate Hydrogen Lyase-Overexpressing Escherichia coli Strains. Applied and Environmental Microbiology. 2005;71(11):6762-6768. doi:10.1128/aem.71.11.6762-6768.2005
Alissandratos A, Kim H-K, Matthews H, Hennessy JE, Philbrook A, Easton CJ. Clostridium carboxidivorans Strain P7T Recombinant Formate Dehydrogenase Catalyzes Reduction of CO2to Formate. Applied and Environmental Microbiology. 2013;79(2):741-744. doi:10.1128/aem.02886-12