Team:Macquarie Australia/Results

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.

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.

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.

Faculty of Science and Engineering,
Macquarie University
Balaclava Road, North Ryde, NSW, 2109, Australia
E7B 350