Difference between revisions of "Team:Macquarie Australia/Results"
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<li>SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).</li> | <li>SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).</li> | ||
<li>Calculated the rate of hydrogen gas production using a Clark electrode which showed 2.5mL of Hydrogen gas was produced per hour.</li> | <li>Calculated the rate of hydrogen gas production using a Clark electrode which showed 2.5mL of Hydrogen gas was produced per hour.</li> | ||
| − | </ul> | + | </ul></p> |
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<br> | <br> | ||
| − | + | <h1>Results</h1> | |
| + | <br> | ||
| + | <h2>Verifying Restriction Enzymes and Antibiotic Resistance</h2> | ||
| + | <br> | ||
| + | <p align="justify"> | ||
| + | The standard restriction enzymes provided by igem (EcoRI, XbaI, SpeI, PstI) were used to digest PCR products to verify they were functional and cut the cellular DNA as expected. An agarose gel electrophoresis (see Fig.1) was performed on digested photosystem II plasmid (psbMZHWK) KODsmart PCR products, as well as chloramphenicol (CAM), ampicillin (AMP) and Kanamycin (KAN) backbones. Enzymes EcoRI (E), PstI (P), XbaI (X) and SpeI (S) were tested. The results show that all digestions cut the psbMZHWK plasmid at the expected ~1000bp mark and appear to be functional in cutsmart buffer. Furthermore, PCR of CAM and AMP was successful, yet Kan was not (see Fig. 1). | ||
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Revision as of 10:43, 29 October 2017
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.
Results
Verifying Restriction Enzymes and Antibiotic Resistance
The standard restriction enzymes provided by igem (EcoRI, XbaI, SpeI, PstI) were used to digest PCR products to verify they were functional and cut the cellular DNA as expected. An agarose gel electrophoresis (see Fig.1) was performed on digested photosystem II plasmid (psbMZHWK) KODsmart PCR products, as well as chloramphenicol (CAM), ampicillin (AMP) and Kanamycin (KAN) backbones. Enzymes EcoRI (E), PstI (P), XbaI (X) and SpeI (S) were tested. The results show that all digestions cut the psbMZHWK plasmid at the expected ~1000bp mark and appear to be functional in cutsmart buffer. Furthermore, PCR of CAM and AMP was successful, yet Kan was not (see Fig. 1).
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