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

 
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<li>We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (<i>fer</i>, <i>hyd1</i>, <i>hydEF</i>, <i>hydG</i>) 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 Biobrick (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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<li>Once induced, we aimed to test the rate of hydrogen gas production in these cells.</li>
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</ul>
 
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<h1>Experimental Design</h1>
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<h2>Summarised Results:</h2>
 
<ul>
 
<ul>
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<li>Analyse, optimise and construct the necessary gBlocks.</li>
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<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>Digest and ligate gblocks into Biobricks.</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>Digest/Double digest in conjunction with sequencing to verify gBlocks.</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>Digest and ligate gBlocks together via standard assembly.</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>Induce plasmid with IPTG for protein expression.</li>
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<li>Successful production of Hydrogen gas. </li>
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<li>Run cell lysate of <i>fer</i> on SDS-PAGE followed by Mass Spectrometry to analyse gel bands.</li>
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<li>Awarded a <b>Gold Medal.</b></li>
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<li>Test hydrogen production using Clark electrode and gas volume measurement experiment.</li>
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<li><b>Awarded Best Energy Project 2017.</b></li>
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</ul>
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<br>
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<h1>Summarised Results:</h1>
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<ul>
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<li>Construction and confirmation of composite parts: hyperlinks to parts registry for fer/hyd1, hydEFG, and Hydrogen Gas Producing Gene Cluster.</li>
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<li>Improvement of previous part, <i>hydG</i>, 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>Confirmed sequencing of parts.</li>
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<li>Confirmed transformation into competent cells.</li>
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<li>Successful assembly of Omega plasmid in the following order: <i>fer-hyd1-hydEFG</i>. PCR and plasmid double digest confirm the presence of these genes at the expected bands (see Fig. 4).</li>
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<li>SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).</li>
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<li>Calculated the rate of hydrogen gas production using a Clark electrode which showed 2.5mL of Hydrogen gas was produced per hour.</li>
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<h1>Results</h1>
 
<h1>Results</h1>
 
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<h2>Verifying Restriction Enzymes and Antibiotic Resistance</h2>
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<h2>Summary of parts</h2>
 
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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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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>
 
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<h3>1. Assembly - <i>FDX/FNR/hyd1</i>, electron transporters to power a hydrogenase</h3>
 
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<p align="justify">
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Following previous verification of PS2 plasmid, the experiment was repeated in an attempt to PCR Kan backbone along with digested CHLP. This agarose gel revealed the same success of functional restriction enzymes cutting CHLP at ~1300bp (see Fig. 2), however Kan PCR remained unsuccessful.
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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>
 
</p>
 
<br>
 
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<h3>2. Assembly – <i>hydEFG</i>, hydrogenase maturation enzymes</h3>
 
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<p align="justify">
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Therefore, all restriction enzymes were used throughout our project as the gels showed they were not contaminated and cut test biobricks PS2 and CHLP as expected. Our project focused on using CAM resistance in our biobricks.  
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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.
 
</p>
 
</p>
 
<br>
 
<br>
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<br>
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<h3>3. Assembly - Hydrogen gas producing gene cluster</h3>
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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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Following successful screening, the functionality of the fer biobrick was confirmed in our project by purifying out the proteins on a Q column (see Fig. 3). The extracted proteins were then observed with a spectrometre at 550 nm. The results from the spectrometre prove that the <i>fer</i> biobrick has functionality as we observed the reduction of cytochrome C by the oxidisation of NADPH to NADP+ (see Fig. 4).
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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.
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Additionally, an SDS-PAGE gel was run to observe the possible bands corresponding to increased fer proteins when induced with IPTG (see Fig. 5).
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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.
 
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<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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<b><i>Figure 1.</i></b> Agarose gel (1%) electrophoresis of single (EcoRI) and double (EcoRI with PstI) digests of parts.
 
<br>
 
<br>
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<h2><i>fer/hyd1</i> Assembly - 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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<br>
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This biobrick was created to ligate a ferredoxin and ferredoxin reductase (FNR), an electron transporter from NADP+ 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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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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<br>
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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> (~1700bp) and <i>fer</i> (~1700bp) with a plasmid vector backbone of ~2000bp (see Fig. 6).
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These gels validate all constructs of the Hydrogen Gas Producing Gene Cluster composite part.
 
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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 Fig. 7).
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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>
 
<br>
 
<br>
 +
<br>
 +
<h2><i>­FDX/FNR</i> characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase</h2>
 
<p align="justify">
 
<p align="justify">
−
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 Fig. 8).
+
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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<br>
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<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 (see Fig. 5), sequencing results confirmed this.
+
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>
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<img src="https://static.igem.org/mediawiki/2017/8/8f/T--Macquarie_Australia--OH22.png" width="390px" height="300px">
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<h2><i>hydEFG</i> Assembly – Hydrogenase Maturation Enzymes</h2>
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<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>
+
−
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.
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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.


GOLD SPONSORS


BRONZE SPONSORS


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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