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Overview

Key achievements of our team include:

• Construction and testing of composite parts: fer/FNR/hyd1, hydEFG, and Hydrogen Gas Producing Gene Cluster under lac promoters.
• Improvement of previous part: hydG.
• Confirmed sequencing of parts.
• Confirmed transformation into competent cells.
• Quantifying hydrogen gas production using a Clark electrode. (Results)
• Modelling of hydrogen gas production. (Model)
• Construction of a prototype. (Entrepreneurship)

fer/FNR – Electron Transporters Ferredoxin and Ferredoxin Reductase

Following successful screening, the functionality of the fer biobrick was confirmed in our project by purifying out the proteins on a Q column (see Fig. 1). The extracted proteins were then observed with a spectrometre at 550 nm. The results from the spectrometre prove that the fer biobrick has functionality as we observed the reduction of cytochrome C by the oxidisation of NADPH to NADP+ (see Fig. 2).
Additionally, an SDS-PAGE gel was run to observe the possible bands corresponding to increased fer proteins when induced with IPTG (see Fig. 3).

Figure 3. Purification of ferredoxin reductase on Q column fractions for samples A, B and C. The intense yellow colouring corresponds with a greater degree of protein purification. Of the 12 fractions taken from each sample, the ones pictured (A10-12, B1, B6-12 and C1) contained purified protein and were used for spectrometry.

Figure 4. Spectrophometre at 550nm of purified ferredoxin reductase (FNR) fraction. The peaks (blue) correspond to the reduction of cytochrome C by the oxidisation of NADPH to NADP+ by FNR. This validates the characterisation of the fer biobrick.

Figure 5. SDS-PAGE gel of induced and purified fer fractions.

fer/hyd1 – Electron Transporters to Hydrogenase

This biobrick was created to ligate a ferredoxin and ferredoxin reductase (fer/FNR), an electron transporter from NADP+ reduction, to a hydrogenase (hyd1) native in C. reinhardtii. The ferredoxin donates electrons to the hydrogenase for the production of hydrogen gas. Successful assembly was proven using single and double digests of PCR products (see Fig. 4) and through sequence verification.

Figure 4. Agarose gel (1%) electrophoresis of single (EcoRI-HF) and double (EcoRI-HF and PstI) digests of fer/hyd1 gene in transformed colony samples A, B, C and D. Samples A (lanes 3-4), B (lanes 4-5) and C (lanes 6-7) are from the same transformed plate. Samples A and B show expected band weights for the single digests (~5400bp) and double digests (~3400bp and ~2000bp) respectively, and were submitted for sequencing confirmation. Band weights in sample C do not correspond with expected band weights and was unsuccessful. Sample D was spun down prior to loading and no band weights are detected. This gel validates the fer/hyd1 Biobrick to the designed constructs.

hydG and hydE/F – Hydrogenase Maturation Enzymes

The hydG biobrick was constructed by the 2016 Macquarie iGEM, however it was found to have a 1bp mutation which appeared to cause a loss of functionality. This year we have corrected this mutation, verified through screening of transformed cells and sequencing, proving we have a functioning maturation enzyme.
This biobrick was combined with the hydEF biobrick to assist in the formation of the H-cluster (active site) in the Hydrogenase, and successful assembly was proven using single and double digests of PCR products (see Fig. 5) and through sequence verification. This composite part leads to the faster assembly of the hydrogenase complex, allowing our ‘fuel’ cell to produce hydrogen gas at an accelerated pace.

Figure 5. Agarose gel (1%) electrophoresis of single (EcoRI-HF) and double (EcoRI-HF and PstI) digests on hydEFG colony samples A, B and C. All three samples displayed the expected band weights of ~7500bp for single digests and ~35500bp with ~2000bp double digests of successful transformation of hydEFG Biobrick with a CAM backbone.

Hydrogen Gas Producing Gene Cluster

The Hydrogen Gas Producing Gene Cluster plasmid is a composite part; the total construct of genes Fer/FNR/Hyd1/HydEFG (see Fig. 4). All promoters are inducible lac promoters with a -35 and -10 consensus sequences of TTTACA and TATGTT respectively. The ribosome binding sites had a consensus sequence of AAGAAGG following the promoter positioning. The Fer gene is a ferredoxin and ferredoxin reductase (FNR) involved in the transportation of electrons which are passed to Hyd1 (Hydrogenase). The HydEFG proteins act as maturation enzymes that aid hydrogenase activation, so that following IPTG induction, the gene cluster will begin to produce hydrogen gas.

Achievements:

• Constructed recombinant Hydrogen Gas Producing Gene Cluster plasmid with Fer/Hyd1/HydEFG under lac promoter.
• Induced IPTG expression of mature hydrogenase.
• Confirmed sequence results for all genes in Hydrogen Gas Producing Gene Cluster plasmid.
• Proved hydrogen gas production using a Clark electrode data.
• __[MDL4] H₂ production in 2 mL of induced DH5a cells at 0.1 OD after __[MDL5] hours against control group of baseline H₂ production (see Fig._)

Fig 4. Agarose gel (1%) electrophoresis of transformed Hydrogen Gas Producing Gene Cluster plasmid with single (S-EcoRI-HF) and double (D-EcoRI-HF and PstI) digests. Lanes 2-9 were performed on the 23/8/17 of 4 sample colonies of Quick cells. Lanes 3-5 and 7-9 (samples B, C, D) display expected band weights of ~8700bp for single digests and ~8700bp with ~2000bp for double digests. Sample A of Quick cells in lanes 2, 6, 13 and 14 did not possess necessary band weights and were discarded. Sample A of commercial cells in lanes 11 and 12 correspond with expected single and double digest band weights. Samples B and C show expected band weights for all single and double digests in both Quick and commercial cells (lanes 15-22). Sample D in commercial cells (lanes 23 and 24) did not possess the expected band weights and were discarded. Sample D in quick cells (lanes 25 and 26) showed the expected band weights for single and double digests.This gel validates the design construct of the HGPGC plasmid.

Testing rate of hydrogen gas production using a Clark electrode

To test the rate of hydrogen gas produced by the Hydrogen Gas Producing Gene Cluster (HGPGC) cells, the hydrogen gas was measured with a Clark electrode. The electrodes measured the hydrogen gas output from a negative control (untransformed DH5a), transformed Fer/Hyd, and two transformed HGPGC cells. The negative control, Fer/Hyd, and one HGPGC cell were induced with IPTG, since the genes are under a lac promoter. Before the induced cells were put into the electrodes, they were grown to the same concentration and induced at the same time. The cultures were then diluted to the same concentration before going into the electrodes.

A hydrogen pop test using the hydrogen gas produced by the the Fer/FNR/Hyd/HydEFG cells!

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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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