Team:Macquarie Australia/Demonstrate


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Overview


Key achievements of our team include:

  • Construction and testing of composite parts: fer/hyd1, hydEFG , and Hydrogen Gas Producing Gene Cluster
  • Improvement of previous part: hyd1G .
  • Confirmed sequencing of parts.
  • Confirmed transformation into competent cells.
  • Quantifying hydrogen gas production using a Clark electrode.
  • Modelling of hydrogen gas production.
  • Construction of a prototype.


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. 1) 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.


Fig 1. 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 ~5500bp with ~2000bp double digests of successful transformation of hydEFG Biobrick with a CAM backbone.

fer/FNR – Electron Transporters Ferredoxin and Ferredoxin Reductase

Functionality of this biobrick were confirmed[MDL1] this year by running a chromatography [MDL2] to purify proteins with addition of NADH+, and run in a spectrophometer to observe the disappearance of the substrate[MDL3] .
The functionality of the Fer/FNR biobrick, containing ferredoxin and ferredoxin reductase were confirmed in our project by purifying out the proteins with the addition of NADH+ (fill in the actual details cause I don’t know what you did). The extracted proteins were then observed with a spectrometer at WHATEVER WAVELENGTH YOU USED to observe the loss of the NADH+(?) substrate. Additionally an SDS-PAGe gel was run and bands corresponding to expected band weights were extracted and analysed using MALDI-TOF Mass Spectroscopy.


Fig 2. 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 (~6800bp) 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.

fer/hyd1 – Electron Transporters to Hydrogenase

This biobrick was created to ligate a ferredoxin and ferredoxin reductase (fer/FNR); 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. 2) and through sequence verification.



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 and respectively. The ribosome binding sites had a consensus sequence of 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 genes act as maturation enzymes that aid hydrogenase activation, so that following IPTG induction, when under anaerobic conditions, the gene cluster with 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.
  • Quantitatively proved hydrogen gas production using a Clark electrode data.
  • __[MDL4] H2 production in 2mL 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.



A hydrogen pop test using the hydrogen gas produced by the the fed/hyd/hydEFG cells!