Aim
- We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (fer, hyd1, hydEF, hydG) from Chlamydomonas reinhardtii.
- Improved gBlock hydG which demonstrated a loss of functionality (2016) due to a point mutation.
- These gBlocks were inserted into one Biobrick (known as the Hydrogen Producing Gene Cluster) and transformed into Escherichia coli with a lac promoter and chloramphenicol resistance.
- Once induced, we aimed to test the rate of hydrogen gas production in these cells.
Experimental Design
- Analyse, optimise and construct the necessary gBlocks.
- Digest and ligate gblocks into Biobricks.
- Digest/Double digest in conjunction with sequencing to verify gBlocks.
- Digest and ligate gBlocks together via standard assembly.
- Induce plasmid with IPTG for protein expression.
- Run cell lysate of fer on SDS-PAGE followed by Mass Spectrometry to analyse gel bands.
- Test hydrogen production using Clark electrode and gas volume measurement experiment.
Summarised Results:
- Construction and confirmation of composite parts: hyperlinks to parts registry for fer/hyd1, hydEFG, and Hydrogen Gas Producing Gene Cluster.
- Improvement of previous part, hydG, to fix point mutation and provide functionality.
- Successful cloning of lac promoters in front of gene constructs.
- Confirmed sequencing of parts.
- Confirmed transformation into competent cells.
- Successful assembly of Omega plasmid in the following order: fer-hyd1-hydEFG. PCR and plasmid double digest confirm the presence of these genes at the expected bands (see Fig. 4).
- SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).
- Calculated the rate of hydrogen gas production using a Clark electrode which showed 2.5mL of Hydrogen gas was produced per hour.
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