Difference between revisions of "Team:UrbanTundra Edmonton/Results"

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            <h1>Results</h1>
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            <h2>The expression of perchlorate dismutase</h2>
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            <p>For the expression of our Pcr construct, we chose to use the iGEM submission vector PSB1C3. Using the Golden Gate ligation method, a single restriction enzyme, BsaI, was used to assemble the gBlocks in the correct order. The BsaI sites, when cut by the restriction enzyme, leave non-complimentary non-palindromic ends. Due to this, the pieces of our construct would anneal in the orientation that we desired: LacIq, PcrA, and PcrA/B.<p>
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            <h3>Transformation of Pcr<h3>
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            <p>The first attempted transformation into DH5α <i>E. coli</i> was unsuccessful. Using a chemical transformation protocol, we were unable to successfully incorporate our plasmid into the competent cells, producing no colony growth on our plates. In discussion with the UAlberta iGEM team, they suggested to try two different things: an electroporation protocol for the transformation of our plasmid into competent cells as well as to use DH10B <i>E. coli</i> as our chassis. In one of our many collaboration efforts with the UAlberta team, they aided us in preforming an electroporation on both DH5 and DH10B <i>E. coli</i>. Fortunately, this transformation yielded colonies for both chassis, albeit only a few.</p>
  
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<img class="sponsors" src="https://static.igem.org/mediawiki/2017/1/1a/Gel_Pcr.png" class="img-responsive" height="400px">
  
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            <h2>Future Steps</h2>
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            <p>To validate the effectiveness of our part, a screening test involving perchlorate would be conducted. As we found out in our research from last year, chlorite is more toxic than perchlorate, even at extremely small concentrations. In other words, it is easier for <i>E. coli</i> to survive in perchlorate than in chlorite. The contrast in toxicity can be used to determine whether our construct was successfully incorporated into our plasmid. We would do this by plating the <i>E. coli</i> that contains our plasmid onto plates that contain the threshold amount of perchlorate along with growth media, which is around 0.1 M. If our part works, the bacteria would produce perchlorate reductase and would convert the perchlorate on the plate into chlorite. Due to chlorite’s toxicity, it would kill the chassis, preventing significant growth on the plate. Essentially, a plate that showed dead E. coli would verify that our part worked.</p>
  
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<h1>Results</h1>
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<p><b>References</b><br>UrbanTundra_Edmonton 2016. “Experimental” <i>2016.igem.org/Team:UrbanTundra_Edmonton.</i> 2016.</p>
  
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<p>Here you can describe the results of your project and your future plans. </p>
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<h5>What should this page contain?</h5>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<h5>Inspiration</h5>
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<p>See how other teams presented their results.</p>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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Latest revision as of 00:36, 21 November 2017

Urban Tundra | Intelligent Innovation

Team
Project
Parts
Safety
Human Practices
Awards

Results

The expression of perchlorate dismutase

For the expression of our Pcr construct, we chose to use the iGEM submission vector PSB1C3. Using the Golden Gate ligation method, a single restriction enzyme, BsaI, was used to assemble the gBlocks in the correct order. The BsaI sites, when cut by the restriction enzyme, leave non-complimentary non-palindromic ends. Due to this, the pieces of our construct would anneal in the orientation that we desired: LacIq, PcrA, and PcrA/B.

Transformation of Pcr

The first attempted transformation into DH5α E. coli was unsuccessful. Using a chemical transformation protocol, we were unable to successfully incorporate our plasmid into the competent cells, producing no colony growth on our plates. In discussion with the UAlberta iGEM team, they suggested to try two different things: an electroporation protocol for the transformation of our plasmid into competent cells as well as to use DH10B E. coli as our chassis. In one of our many collaboration efforts with the UAlberta team, they aided us in preforming an electroporation on both DH5 and DH10B E. coli. Fortunately, this transformation yielded colonies for both chassis, albeit only a few.

Future Steps

To validate the effectiveness of our part, a screening test involving perchlorate would be conducted. As we found out in our research from last year, chlorite is more toxic than perchlorate, even at extremely small concentrations. In other words, it is easier for E. coli to survive in perchlorate than in chlorite. The contrast in toxicity can be used to determine whether our construct was successfully incorporated into our plasmid. We would do this by plating the E. coli that contains our plasmid onto plates that contain the threshold amount of perchlorate along with growth media, which is around 0.1 M. If our part works, the bacteria would produce perchlorate reductase and would convert the perchlorate on the plate into chlorite. Due to chlorite’s toxicity, it would kill the chassis, preventing significant growth on the plate. Essentially, a plate that showed dead E. coli would verify that our part worked.

References
UrbanTundra_Edmonton 2016. “Experimental” 2016.igem.org/Team:UrbanTundra_Edmonton. 2016.

View Project