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Hydrogen Gas Producing Gene Cluster - HGPC

Our Validation Work

We validated our Hydrogen Gas Producing Gene Cluster (HGPGC) part in two ways. We measured the volume of total gas produced and we measured 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. Hydrogen production was measured using a technique which 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 (Fig. 3). A maximum rate of gas production for induced HGPGC cells was 3.39 mM H2 hr-1 (or 6.08 mL hr-1).

Fig 3. Gas production over time for the three cultures: HGPGC cells, Fer/Hyd and non-transformed DH5α. 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 induced HGPGC cells was 0.11 mM H2 hr-1 (or 4.7 mL H2 h-1). Fig 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

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

This validation from Singapore helped clarify that all of the genes in our Hydrogen Gas Producing Gene Cluster were expressing. Albeit, with HydG expressing less for some unknown reason.

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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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