Team:Macquarie Australia/Results
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 Biobrick (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: 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).
Results
Summary of Parts
All composite parts underwent single (EcoRI) and double (EcoRI with PstI) digests followed by agarose gel (1%) electrophoresis to summarize and validate the successful construction of the parts comprising the Hydrogen Gene Producing Cluster (see Figure 1).
Figure 1. Agarose gel (1%) electrophoresis of single (EcoRI) and double (Eco-RI with PstI) digests of parts.
Left: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show single (~10,700 bp) and double (~8700 bp with ~2000 bp) digests respectively of the composite Hydrogen Gas Producing Gene Cluster plasmid (HGPGC). Lanes 4 and 5 show single (~7400 bp) and double (faint ~5400 bp with ~2000 bp) digests of hydEFG. Lanes 6 and 7 show single (~5400 bp) and double digests (~3400 bp with ~2000 bp) of fer/hyd1.
Right: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show double digests (~1900 bp with ~2000 bp) and single digest (~3900 bp) of hydG.
These gels validate all constructs of the Hydrogen Gas Producing Gene Cluster.
fer/FNR characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase
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fer/hyd1 Assembly - 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 Fig. 6).
Figure 6. Agarose gel (1%) electrophoresis. Lanes 7 and 14 show 1kb marker. Lanes 1, 3, 5, show single digest of fer plasmid using Eco-RI (E) at ~2700 bp. Lanes 2, 4 and 6 show double digest of fer using Eco-RI and PstI (E+P) with bands at ~1700 bp and ~2000 bp. Lanes 8, 10 and 12 show hyd1 single digest with E (~3700bp). Lanes 9, 11 and 13 show double digest of hyd1 using E+P (~1700 bp and ~2000 bp).
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 Fig. 7).
Figure 7. Agarose gel (1%) electrophoresis of single and double digests using Eco-RI (E) and PstI (P) in fer/hyd1 gene in transformed colony samples A, B, C and D. Lane 1 contains a 1kb ladder. 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 (~5400bp) and double digests (~3400 bp and 2000 bp) respectively, and were submitted for sequencing confirmation. Band weights in sample C do not correspond with expected band weights and were unsuccessful. Sample D was spun down prior to loading and no band weights were detected. This gel validates the fer/hyd1 Biobrick to the designed constructs in samples A and B.
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 Fig. 8).
Figure 8. Agarose gel (1%) electrophoresis of Biobrick fer/hyd after having the backbone swapped to CAM resistance using digests using Eco-RI (E) and PstI (P). Single (E) and double (E+P) digests were performed. The bands of the single digests correspond to the expected size (~5300 bp) as well as the bands of the double digests (~3500 bp and ~2000 bp). These bands correspond with the expected weights of fer/hyd1 in CAM.
In summary the biobricks fer and hyd1 were successfully ligated together (see Fig. 5), sequencing results confirmed this.
hydEFG Assembly – 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 Fig. 9) and sequencing means this plasmid will allow the faster assembly of the hydrogenase complex, in turn allowing our cell to produce hydrogen gas sooner.
Figure 9. Agarose gel (1%) electrophoresis of single (E) and double (E+P) digests on colony samples A, B and C. All three samples display expected band weights of ~7500bp for single digests and ~5500bp with ~2000bp double digests. This gel indicates successful ligation of hydG and hydEF biobricks and validates the hydEFG biobrick.
Hydrogen gas producing gene cluster Assembly
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 Fig. 10). 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.
Figure 10. 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 ~10,700bp for single digests and ~8700 bp with ~2000 bp 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.
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.
Demonstrating HGPGC produces hydrogen gas
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