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+ | To quantify the 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 <i>lac</i> 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. | ||
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<center> <img src="https://static.igem.org/mediawiki/2017/c/c7/H2Decay.png" width="239px" height="240px"> </center> | <center> <img src="https://static.igem.org/mediawiki/2017/c/c7/H2Decay.png" width="239px" height="240px"> </center> | ||
Revision as of 07:44, 26 October 2017
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
Achievements:
Quantifying hydrogen gas production using a Clark electrode
To quantify the 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.