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Hydrogen Gas Producing Gene Cluster - HGPC

Validation, the confirmation by examination and provision of evidence showing functionality of intended use, is an important step that should occur after scientific research has resulted in the development or discovery a new product.

Our Validation Work

We validated our Hydrogen Gas Producing Gene Cluster (HGPGC) part in two ways:

1. We measured the volume of total gas produced and the hydrogen produced in solution-
• Total gas was collected and measured using an apparatus involving upturned measuring cylinders partially submerged in water and linked to cultures via tubing.
• In this way we could measure the total volume of gas produced over time for three cultures: HGPGC cells, Fer/Hyd cells and original non-transformed DH5α.
2. Hydrogen production was measured using Clark electrodes-
• This technique reverses polarity of a Clark electrode which is typically used to measure oxygen in solution.
• In this way we were able to collect hydrogen production data across four cultures: HGPC cells (both induced and uninduced), Fer/Hyd cells and original non-transformed DH5α.
• Overall production of gas was much higher for our HGPGC cells (Figure 3). A maximum rate of gas production for 2 mL induced HGPGC cells was 2.37 mL H₂/hr/L.

Figure 1. Set-up of upturned cylinder experiments, used to measure volume of gas produced over time for the three cultures: HGPGC cells, Fer/Hyd and non-transformed DH5α.

Figure 2. Clark electrode measurement set-up.

The hydrogen production in the Clark electrodes showed that production of hydrogen was not only higher in the induced HGPGC cells, compared to other cultures, but that production was sustained over a longer time period. The maximum rate of hydrogen production for 80 mL induced HGPGC cells in the upturned cylinder experiments, was 7.0 mL H₂/hr/L (Figure 3., Figure 4.). It can be seen that for replicate 2 (Figure 3) HGPGC did not produce as much gas. Both replicates used the same original mature culture. The culture had been kept refrigerated for approximately three weeks when data was collected for replicate 2, which indicates optimal production may be found with a fresh culture. This information is valuable feeding into our prototype and commercial model which provides at home hydrogen production.

Figure 3. Volume of gas produced from upturned cylinder experiments measured over time for the three cultures: HGPGC cells, Fer/Hyd and non-transformed DH5α.

Figure 4. Clark electrode data showing overall hydrogen production over time for the four cultures HGPGC induced and uninduced, Fer/Hyd and non-transformed DH5α.

These experiments indicate that not only does our part realise the capability of producing hydrogen but, by comparison with Fer/Hyd alone, we can see that the maturation enzymes included in HGPGC cells are contributing by increasing the rate, volume and longevity of hydrogen production. HGPGC also exceeds any hydrogen production from the E. coli endogenous hydrogenase.

Sequence Validation

We sent our Hydrogen Gas Producing Gene Cluster (HGPGC) to be sequenced and were very happy to have the results come back positive! Please see the parts page for the full sequence.

NTU Singapore's Validation

This year again, we sought assistance from team NTU Singapore. They helped us to evaluate and validate the gene expression level of each of the genes in our Hydrogen Gas Producing Gene Cluster, by using RT-qPCR.

They quote - "IPTG and 20mM of glucose was used to induce the gene expression. Comparison of 1mM IPTG and 20mM IPTG was made and it was found that 1mM IPTG has higher gene expression level. It is worth to notice that the RT-PCR efficiency might be different for different genes due to their size of PCR products and the formula used above is assuming the PCR efficiency to be 100% that eventually resulted in 2 copies of products." NTU Singapore

Figure 5. qPCR results of hydrogenase gene expression relative to Chloramphenicol with different concentrations of IPTG.

This validation from Singapore helped clarify that all of the genes in our Hydrogen Gas Producing Gene Cluster were expressed.

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LOCATION Faculty of Science and Engineering, Macquarie University Balaclava Road, North Ryde, NSW, 2109, Australia E7B 350 CONTACT US Email: macquarie.australia@gmail.com

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