Aim

• We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (Fdx, FNR, 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 composite part (known as the Hydrogen Producing Gene Cluster) and transformed into Escherichia coli with a lac promoter and chloramphenicol resistance.

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.
• Test hydrogen production using Clark electrode and gas volume measurement experiment.

Summarised Results:

• Construction and confirmation of composite parts: fer/FNR/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.
• SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase.
• Successful assembly of composite part - HGPGC in the following order: fer-FNR-hyd1-hydEFG.

Results

Summary of parts

All composite parts underwent single (EcoRI) and double (EcoRI with PstI) digests followed by agarose gel (1%) electrophoresis to summarise and validate the successful construction of the parts comprising the Hydrogen Gene Producing Gene Cluster (HGPGC) (see Figure 1). All parts are also sequenced confirmed.

Assembly - Hydrogen gas producing gene cluster

With sequencing of the biobricks fer/hyd1 and hydEFG confirmed, all that remained was a final assembly. The Hydrogen Gas Producing Gene Cluster plasmid is a composite part; the total construct of genes fer/FNR/hyd1/hydEFG (see Figure 1). All promoters are inducible lac promoters with a -35 and -10 consensus sequences of TTTACA and TATGTT respectively. The ribosome binding sites had a sequence of aagaagg following the promoter positioning.

The fer genes are 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 will begin to produce hydrogen gas.

Assembly - fer/hyd1, electron transporters to power a hydrogenase

This biobrick was created to ligate a ferredoxin and ferredoxin reductase (FNR), an electron transporter from NADP⁺ reduction, to a hydrogenase native in C. reinhardtii. The ferredoxin donates electrons to the hydrogenase for the production of hydrogen gas.
The biobricks fer-FNR (fer –ferredoxin and ferredoxin reductase) and hyd1 ([FeFe] hydrogenase) were screened prior to their assembly by single and double digestions with E and E+P enzymes. Digests were run on agarose gel (1%) and showed appropriate sites were cut in hyd1 (~1700 bp) and fer (~1700 bp) with a plasmid vector backbone of ~2000 bp (see Notebook, Week 7).

Standard assembly of verified biobricks fer and hyd1 was performed with CAM or AMP resistance. The ligated biobricks were transformed into competent cells and plated onto CAM/AMP plates respectively. Colonies grew, which were further incubated, miniprepped and screened (see Notebook, Week 8).

Following the validation of the fer/hyd1 biobrick by sequencing, the backbone was swapped to CAM resistance as all other biobricks we created were using this type of antibiotic resistance. The fer/hyd1 backbone was successfully swapped to CAM (see Figure 1).

In summary the biobricks fer and hyd1 were successfully ligated together (Figure 1) and sequencing results confirmed this.

Assembly – hydEFG, 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 and following biobrick creation with transformation into competent DH5α cells, the sequenced results prove we have a functioning maturation enzyme.
This biobrick was ligated with biobrick hydEF to assist in the formation of the H-cluster in the Hydrogenase. Confirmed transformation into competent cells (see Figure 2) and sequencing means this plasmid will allow the faster assembly of the hydrogenase complex, in turn allowing our cell to produce hydrogen gas sooner.

­fer/FNR characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase

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Demonstrating HGPGC produces hydrogen gas

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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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