Difference between revisions of "Team:Macquarie Australia/Results"

 
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<p align="justify">
 
<p align="justify">
 
<ul>
 
<ul>
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<li>We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (Fdx, FNR, Hyd1, HydEF, HydG) from <i>Chlamydomonas reinhardtii</i>. </li>
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<li>We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (FDX, FNR, Hyd1, HydEF, HydG) from <i>Chlamydomonas reinhardtii</i>. </li>
 
<li>Improved gBlock <i>hydG</i> which demonstrated a loss of functionality (2016) due to a point mutation.</li>
 
<li>Improved gBlock <i>hydG</i> which demonstrated a loss of functionality (2016) due to a point mutation.</li>
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<li>These gBlocks were inserted into one composite part (known as the Hydrogen Producing Gene Cluster) and transformed into <i>Escherichia coli</i> with a <i>lac</i> promoter and chloramphenicol resistance. </li>
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<li>Wanted the gBlocks to be inserted into one composite part (known as the Hydrogen Producing Gene Cluster) and transformed into <i>Escherichia coli</i> with <i>lac</i> promoters and chloramphenicol resistance. </li>
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</ul>
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<br>
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<br>
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<h2>Experimental Design</h2>
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<ul>
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<li>Analyse, optimise and construct the necessary gBlocks.</li>
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<li>Digest and ligate gblocks into Biobricks.</li>
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<li>Digest/Double digest in conjunction with sequencing to verify gBlocks.</li>
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<li>Digest and ligate gBlocks together via standard assembly.</li>
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<li>Induce plasmid with IPTG for protein expression.</li>
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<li>Run cell lysate of Fer on SDS-PAGE.</li>
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<li>Test hydrogen production using Clark electrode and gas volume measurement experiment.</li>
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</ul>
 
</ul>
 
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<h2>Summarised Results:</h2>
 
<h2>Summarised Results:</h2>
 
<ul>
 
<ul>
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<li>Construction and confirmation of composite parts: <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_S05396> <i>fer/FNR/hyd1</i></a>, <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300000> <i>hydEFG</i></a>, and <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300001> Hydrogen Gas Producing Gene Cluster</a></li>
 
 
<li>Improvement of previous part, <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300002><i>hydG</i></a>, to fix point mutation and provide functionality.</li>
 
<li>Improvement of previous part, <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300002><i>hydG</i></a>, to fix point mutation and provide functionality.</li>
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<li>Successful cloning of <i>lac</i> promoters in front of gene constructs.</li>
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<li>Construction and sequence confirmation of composite parts: <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_S05396> <i>fer/FNR/hyd1</i></a>, <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300000> <i>hydEFG</i></a>, and <a href =http://parts.igem.org/wiki/index.php?title=Part:BBa_K2300001> Hydrogen Gas Producing Gene Cluster</a></li>
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<li>Confirmed sequencing of parts.</li>
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<li>SDS-PAGE and enzymatic assay of induced protein expression of ferredoxin and ferredoxin reductase.</li>
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<li>Confirmed transformation into competent cells.</li>
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<li>Successful assembly of composite part - HGPGC in the following order: <i>fer-FNR-hyd1-hydEFG</i>. </li>
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<li>SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).</li>
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<li>Successful production of Hydrogen gas. </li>
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<li>Successful assembly of composite part - HGPGC in the following order: <i>fer-hyd1-hydEFG</i>
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<li>Awarded a <b>Gold Medal.</b></li>
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<li><b>Awarded Best Energy Project 2017.</b></li>
 
  </ul></p>
 
  </ul></p>
 
<br>
 
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<h2>Summary of parts</h2>
 
<h2>Summary of parts</h2>
 
<p align="justify">
 
<p align="justify">
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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).  
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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.
 
</p>
 
</p>
 
<br>
 
<br>
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<center><img src="https://static.igem.org/mediawiki/2017/2/2f/T--Macquarie_Australia--finalgel.png" width="500px" height="409px"> </center>
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<h3>1. Assembly - <i>FDX/FNR/hyd1</i>, electron transporters to power a hydrogenase</h3>
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<center> <p align="justify" style="width:500px;word-wrap:break-word">
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<p align="justify">
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<b><i>Figure 1.</i></b> Agarose gel (1%) electrophoresis of single (EcoRI) and double (Eco-RI with PstI) digests of parts.
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This biobrick was created to ligate a ferredoxin (FDX) and ferredoxin reductase (FNR), an electron transporter from NADP<sup>+</sup> reduction, to a hydrogenase native in <i>C. reinhardtii</i>. The ferredoxin donates electrons to the hydrogenase for the production of hydrogen gas. Standard assembly of verified biobricks <i>FDX/FNR</i> and <i>hyd1</i> was performed in a CAM backbone (see <a href =https://2017.igem.org/Team:Macquarie_Australia/Notebook>Notebook</a>, Weeks 7, 8). The biobricks <i>FDX/FNR</i> and <i>hyd1</i> were successfully ligated together (Figure 1) and sequencing results confirmed this.</b>
 +
</p>
 
<br>
 
<br>
−
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.
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<h3>2. Assembly – <i>hydEFG</i>, hydrogenase maturation enzymes</h3>
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<br>
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<p align="justify">
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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 <i>hydG</i>.
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The <i>hydG</i> 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 <i>hydEF</i> to assist in the formation of the H-cluster in the Hydrogenase.
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<br>
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These gels validate all constructs of the Hydrogen Gas Producing Gene Cluster composite part.
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</p>
 
</p>
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</center>
 
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<br>
 
 
<br>
 
<br>
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<h2>Assembly - Hydrogen gas producing gene cluster</h2>
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<h3>3. Assembly - Hydrogen gas producing gene cluster</h3>
 
<p align="justify">
 
<p align="justify">
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With sequencing of the biobricks <i>fer/hyd1</i> and <i>hydEFG</i> confirmed, all that remained was a final assembly. The Hydrogen Gas Producing Gene Cluster plasmid is a composite part; the total construct of genes <i>fer/FNR/hyd1/hydEFG</i> (see Figure 1). All promoters are inducible <i>lac</i> 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.
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With sequencing of the biobricks <i>FDX/FNR-hyd1</i> and <i>hydEFG</i> confirmed, all that remained was a final assembly. The Hydrogen Gas Producing Gene Cluster plasmid is a composite part; the total construct of genes <i>FDX/FNR/hyd1/hydEFG</i> (see Figure 1). All promoters are inducible <i>lac</i> 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.
 
<br>
 
<br>
 
<br>
 
<br>
−
The <i>fer</i> genes are a ferredoxin and ferredoxin reductase (FNR) involved in the transportation of electrons which are passed to <i>hyd1</i> (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.
+
The <i>fer</i> genes are a ferredoxin (FDX) and ferredoxin reductase (FNR) involved in the transportation of electrons which are passed to <i>hyd1</i> (Hydrogenase). The <i>hydEFG</i> 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.
 
</p>
 
</p>
 
<br>
 
<br>
 +
<center><img src="https://static.igem.org/mediawiki/2017/2/2f/T--Macquarie_Australia--finalgel.png" width="731px" height="600px"> </center>
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<center> <p align="justify" style="width:731px;word-wrap:break-word">
 +
<b><i>Figure 1.</i></b> Agarose gel (1%) electrophoresis of single (EcoRI) and double (EcoRI with PstI) digests of parts.
 
<br>
 
<br>
−
<h2>Assembly - <i>fer/hyd1</i>, electron transporters to power a hydrogenase</h2>
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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 <i>hydEFG</i>. Lanes 6 and 7 show single (~5400 bp) and double digests (~3400 bp with ~2000 bp) of <i>fer/hyd1</i>.
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<p align="justify">
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This biobrick was created to ligate a ferredoxin and ferredoxin reductase (FNR), an electron transporter from NADP<sup>+</sup> reduction, to a hydrogenase native in <i>C. reinhardtii</i>. The ferredoxin donates electrons to the hydrogenase for the production of hydrogen gas.
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<br>
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The biobricks <i>fer-FNR</i> (<i>fer –ferredoxin</i> and <i>ferredoxin reductase</i>) and <i>hyd1</i> ([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 <i>hyd1</i> (~1700 bp) and <i>fer</i> (~1700 bp) with a plasmid vector backbone of ~2000 bp (see Figure 2).
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</p>
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<br>
 
<br>
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<center><img src="https://static.igem.org/mediawiki/2017/7/76/T--Macquarie_Australia--ferhyd1.png" width="500px" height="385px"> </center>
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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 <i>hydG</i>.
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<center> <p align="justify" style="width:500px;word-wrap:break-word">
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<br>
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<b><i>Figure 2.</i></b> 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 <i>hyd1</i> using E+P (~1700 bp and ~2000 bp).
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These gels validate all constructs of the Hydrogen Gas Producing Gene Cluster composite part.
 
</p>
 
</p>
 
</center>
 
</center>
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<br>
 
<br>
 
<p align="justify">
 
<p align="justify">
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Standard assembly of verified biobricks <i>fer</i> and <i>hyd1</i> 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 Figure 3).
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For more information on our validation, <a href =https://2017.igem.org/Team:Macquarie_Australia/Validation>go here</a>.
 
</p>
 
</p>
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<br>
 
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<center><img src="https://static.igem.org/mediawiki/2017/d/d7/T--Macquarie_Australia--ferhyd2.png" width="428px" height="599px"> </center>
 
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<center> <p align="justify" style="width:428px;word-wrap:break-word">
 
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<b><i>Figure 3.</i></b> 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.
 
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</p>
 
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</center>
 
 
<br>
 
<br>
 
<br>
 
<br>
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<h2><i>­FDX/FNR</i> characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase</h2>
 
<p align="justify">
 
<p align="justify">
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Following the validation of the <i>fer/hyd1</i> biobrick by sequencing, the backbone was swapped to CAM resistance as all other biobricks we created were using this type of antibiotic resistance. The <i>fer/hyd1</i> backbone was successfully swapped to CAM (see Figure 4).
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More information on our improvement of this part is located <a href =https://2017.igem.org/Team:Macquarie_Australia/Improve>here</a>.
 
</p>
 
</p>
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<img src="https://static.igem.org/mediawiki/2017/e/ee/T--Macquarie_Australia--improvefig3.png" width="360px" height="300px"> </center>
 
<br>
 
<br>
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<center><img src="https://static.igem.org/mediawiki/2017/f/f7/T--Macquarie_Australia--ferhyd3.png" width="500px" height="502px"> </center>
 
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<center> <p align="justify" style="width:500px;word-wrap:break-word">
 
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<b><i>Figure 4.</i></b> 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 <i>fer/hyd1</i> in CAM.
 
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</p>
 
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</center>
 
 
<br>
 
<br>
 
<br>
 
<br>
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<h2> Demonstrating HGPGC produces hydrogen gas</h2>
 
<p align="justify">
 
<p align="justify">
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In summary the biobricks <i>fer</i> and <i>hyd1</i> were successfully ligated together (Figure 4) and sequencing results confirmed this.
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More information on how we demonstrated the functioning of our parts is located <a href =https://2017.igem.org/Team:Macquarie_Australia/Demonstrate>here</a>.
 
</p>
 
</p>
 +
<img src="https://static.igem.org/mediawiki/2017/8/8f/T--Macquarie_Australia--OH22.png" width="390px" height="300px">
 
<br>
 
<br>
 
<br>
 
<br>
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<h2> Assembly – <i>hydEFG</i>, hydrogenase maturation enzymes</h2>
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<br>
 +
<h2> Modeling of HGPGC hydrogen gas production</h2>
 
<p align="justify">
 
<p align="justify">
−
The <i>hydG</i> 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.
+
For our modeling of the functioning of our composite part, <a href =https://2017.igem.org/Team:Macquarie_Australia/Model>go here</a>.
 
<br>
 
<br>
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This biobrick was ligated with biobrick <i>hydEF</i> to assist in the formation of the H-cluster in the Hydrogenase. Confirmed transformation into competent cells (see Figure 5) and sequencing means this plasmid will allow the faster assembly of the hydrogenase complex, in turn allowing our cell to produce hydrogen gas sooner.
 
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</p>
 
 
<br>
 
<br>
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<center><img src="https://static.igem.org/mediawiki/2017/2/21/T--Macquarie_Australia--PrettyGel.png" width="500px" height="450px"> </center>
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<img src="https://static.igem.org/mediawiki/2017/b/bd/Screen_Shot_2017-11-01_at_11.33.48_am.png">
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<center> <p align="justify" style="width:500px;word-wrap:break-word">
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<b><i>Figure 5.</i></b> 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.</p>
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</center>
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<h2><i>­fer/FNR</i> characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase</h2>
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<p align="justify">
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For more information (<a href =https://2017.igem.org/Team:Macquarie_Australia/Improve>go here</a>).
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</p>
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<br>
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<h2> Demonstrating HGPGC produces hydrogen gas</h2>
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<p align="justify">
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For more information (<a href =https://2017.igem.org/Team:Macquarie_Australia/Demonstrate>go here</a>).
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{{Team:Macquarie_Australia/Templates/Footer}}

Latest revision as of 22:44, 24 November 2017



menubanner

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.
  • Wanted the gBlocks to be inserted into one composite part (known as the Hydrogen Producing Gene Cluster) and transformed into Escherichia coli with lac promoters and chloramphenicol resistance.


Summarised Results:

  • Improvement of previous part, hydG, to fix point mutation and provide functionality.
  • Construction and sequence confirmation of composite parts: fer/FNR/hyd1, hydEFG, and Hydrogen Gas Producing Gene Cluster
  • SDS-PAGE and enzymatic assay of induced protein expression of ferredoxin and ferredoxin reductase.
  • Successful assembly of composite part - HGPGC in the following order: fer-FNR-hyd1-hydEFG.
  • Successful production of Hydrogen gas.
  • Awarded a Gold Medal.
  • Awarded Best Energy Project 2017.



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.


1. Assembly - FDX/FNR/hyd1, electron transporters to power a hydrogenase

This biobrick was created to ligate a ferredoxin (FDX) 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. Standard assembly of verified biobricks FDX/FNR and hyd1 was performed in a CAM backbone (see Notebook, Weeks 7, 8). The biobricks FDX/FNR and hyd1 were successfully ligated together (Figure 1) and sequencing results confirmed this.


2. 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.


3. Assembly - Hydrogen gas producing gene cluster

With sequencing of the biobricks FDX/FNR-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 FDX/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 (FDX) 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.


Figure 1. Agarose gel (1%) electrophoresis of single (EcoRI) and double (EcoRI 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 composite part.



For more information on our validation, go here.



­FDX/FNR characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase

More information on our improvement of this part is located here.




Demonstrating HGPGC produces hydrogen gas

More information on how we demonstrated the functioning of our parts is located here.




Modeling of HGPGC hydrogen gas production

For our modeling of the functioning of our composite part, go here.


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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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