Difference between revisions of "Team:Toronto/Parts"

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<div class="section">
 
<div class="section">
 
<div class="container header" id="dark-cyan">
 
<div class="container header" id="dark-cyan">
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<h1>Safety</h1>
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<h1>Parts</h1>
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<p>Safety is the number one priority</p>
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</div>
 
</div>
 
</div>
 
</div>
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<div class="subsection">
 
<div class="subsection">
  
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<h2>General Lab Safety</h2>
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<h2>Submitted Parts</h2>
 +
 
 +
<!-- Figure start -->
 +
<figure>
 +
<div class="figures">
 +
<table>
 +
  <tr>
 +
      <td>pSB1C3</td>
 +
      <td>Registry number </td>
 +
      <td>Type</td>
 +
      <td>Length (bp)</td>
 +
      <td>Function </td>
 +
      <td>Assembly Method</td>
 +
      <td>UNS</td>
 +
  </tr>
 +
  <tr>
 +
      <td>LacILOV-mCherry </td>
 +
      <td>BBa_K2469003</td>
 +
      <td>Composite </td>
 +
      <td>1615</td>
 +
      <td>Light regulated reporter </td>
 +
      <td>Digestion and ligation </td>
 +
      <td>2, 3</td>
 +
  </tr>
 +
  <tr>
 +
      <td>HIS-AcrIIA4</td>
 +
      <td>BBa_K2469002</td>
 +
      <td>Basic</td>
 +
      <td>398</td>
 +
      <td>Inhibits the function of S. pyogenes Cas9 and dCas9.</td>
 +
      <td>Gibson assembly of amplified AcrIIA4 from G-block and amplified pSB1C3 backbone from LacILOV-mCherry Construct in pSB1C3</td>
 +
      <td>2, 3</td>
 +
  </tr>
 +
  <tr>
 +
      <td>LacILOV</td>
 +
      <td>BBa_K2469001</td>
 +
      <td>Basic</td>
 +
      <td>728</td>
 +
      <td>A fusion protein of the light, oxygen and voltage-sensitive domain of the Avena Sativa phototropin 1 (LOV2) and the LacI domain. This transcriptional regulator represses promoters under control of LacI. In the presence of blue light this protein will act as a repressor, and in its absence the protein will dissociate from the promoter </td>
 +
      <td>Gibson assembly of amplified LacILOV part from LacILOV-mCherry construct in pKDL071 and pSB1C3 backbone from LacILOV-mCherrt construct in pSCB1C3</td>
 +
      <td>2, 3</td>
 +
  </tr>
 +
  <tr>
 +
      <td>reporter construct </td>
 +
      <td>BBa_K2469004</td>
 +
      <td>Composite </td>
 +
      <td>3253</td>
 +
      <td>Light regulated reporter switch, alternating between mCherry and YFP flourescence </td>
 +
      <td>Gibson assembly of amplified reporter construct out of pkDL071 and amplified pSB1C3 backbone from the LacILOV-mCherry construct in pSB1C3 </td>
 +
      <td>2, 3</td>
 +
  </tr>
 +
</table>
 +
</div>
 +
<figcaption>Figure 1.1: Parts</figcaption>
 +
</figure>
 +
<!-- figure end -->
  
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<p>Before starting the project, our team completed the biosafety and the biosecurity trainings provided by the University of Toronto Biosafety office, and our project supervisor, Dr. Radhakrishnan Mahadevan. The training modules completed include lab access and rules, biosafety levels and equipment, emergency procedures etc.</p>
 
−
<p>Overall, our team adhered to the Canadian Biosafety Standards and Guidelines.<sup><a href="#ref1">[1]</a></sup> The biosafety of our lab was overseen by the University's Institutional Biosafety Committee, with administrative and technical support provided by the Biosafety Team in the Office of Environmental Health and Safety.<sup><a href="#ref2">[2]</a></sup></p>
 
 
</div>
 
</div>
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<!-- subsection 3 END -->
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<!-- subsection 1 END -->
 +
 
 +
 
 +
<!-- subsection 2 -->
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<div class="subsection">
 +
 
 +
<h2>Other Parts</h2>
 +
 
 +
<!-- Figure start -->
 +
<figure>
 +
<div class="figures">
 +
<table>
 +
  <tr>
 +
      <td>pKDLO71</td>
 +
      <td>Type </td>
 +
      <td>Length</td>
 +
      <td>Function</td>
 +
      <td>Assembly Method</td>
 +
      <td>UNS</td>
 +
  </tr>
 +
  <tr>
 +
      <td>LacILOV-mCherry</td>
 +
      <td>Composite </td>
 +
      <td>1615</td>
 +
      <td>Light regulated reporter </td>
 +
      <td>Gibson g-Block + Backbone with UNS</td>
 +
      <td>2, 3</td>
 +
  </tr>
 +
  <tr>
 +
      <td>reporter mCherry</td>
 +
      <td></td>
 +
      <td></td>
 +
      <td>reporter construct, consisting of the CI repressor (with LVA degradation tag) and mCherry under the control of the Ptrc-2 promoter; used as a negative control in the sgRNA assay </td>
 +
      <td>Gibson g-Block + Backbone with UNS</td>
 +
      <td>2, 4</td>
 +
  </tr>
 +
  <tr>
 +
      <td>reporter mCherry + sgRNA </td>
 +
      <td></td>
 +
      <td></td>
 +
      <td>Half of the full construct, to test the function of the sgRNA (ensure it is targetting the correct sequence) </td>
 +
      <td>Gibson assembly of sgRNA g-Block into reporter mCherry, with UNS 5 extension on backbone </td>
 +
      <td>4, 5</td>
 +
  </tr>
 +
  <tr>
 +
      <td>reporter mCherry + sgRNA+ AcrIIA4 </td>
 +
      <td></td>
 +
      <td></td>
 +
      <td>Incorporation of the Anti-CRISPR protein into the sgRNA construct to have both expressed simultaneously and test the function of the Anti-CRISPR </td>
 +
      <td>Gibson assemly of AcrIIA4 g-block into reporter mCherry + sgRNA, with UNS 6 extension on backbone </td>
 +
      <td>4, 6</td>
 +
  </tr>
 +
  <tr>
 +
      <td>reporter construct </td>
 +
      <td>Composite </td>
 +
      <td>3253</td>
 +
      <td>Light regulated reporter switch, alternating between mCherry and YFP flourescence </td>
 +
      <td>Gibson assembly of reporter YFP (LacILOV + YFP) into reporter mCherry with UNS 3 extension on backbone </td>
 +
      <td>3, 2</td>
 +
  </tr>
 +
  <tr>
 +
      <td>full construct </td>
 +
      <td></td>
 +
      <td></td>
 +
      <td>Light-regulated switch controlling the activity of the CRISPR-Cas9 system. </td>
 +
      <td>Gibson assembly of AcrIIA4 subpart (LacILOV + AcrIIA4 + YFP) into reporter mCherry + sgRNA, with UNS 3 extension on bacbone  </td>
 +
      <td>3, 6</td>
 +
  </tr>
 +
</table>
 +
</div>
 +
<figcaption>Figure 1.1: Other parts</figcaption>
 +
</figure>
 +
<!-- figure end -->
 +
 
 +
</div>
 +
<!-- subsection 2 END -->
 +
 
  
 
<hr>
 
<hr>

Revision as of 00:40, 1 November 2017

Parts

Submitted Parts

pSB1C3 Registry number Type Length (bp) Function Assembly Method UNS
LacILOV-mCherry BBa_K2469003 Composite 1615 Light regulated reporter Digestion and ligation 2, 3
HIS-AcrIIA4 BBa_K2469002 Basic 398 Inhibits the function of S. pyogenes Cas9 and dCas9. Gibson assembly of amplified AcrIIA4 from G-block and amplified pSB1C3 backbone from LacILOV-mCherry Construct in pSB1C3 2, 3
LacILOV BBa_K2469001 Basic 728 A fusion protein of the light, oxygen and voltage-sensitive domain of the Avena Sativa phototropin 1 (LOV2) and the LacI domain. This transcriptional regulator represses promoters under control of LacI. In the presence of blue light this protein will act as a repressor, and in its absence the protein will dissociate from the promoter Gibson assembly of amplified LacILOV part from LacILOV-mCherry construct in pKDL071 and pSB1C3 backbone from LacILOV-mCherrt construct in pSCB1C3 2, 3
reporter construct BBa_K2469004 Composite 3253 Light regulated reporter switch, alternating between mCherry and YFP flourescence Gibson assembly of amplified reporter construct out of pkDL071 and amplified pSB1C3 backbone from the LacILOV-mCherry construct in pSB1C3 2, 3
Figure 1.1: Parts

Other Parts

pKDLO71 Type Length Function Assembly Method UNS
LacILOV-mCherry Composite 1615 Light regulated reporter Gibson g-Block + Backbone with UNS 2, 3
reporter mCherry reporter construct, consisting of the CI repressor (with LVA degradation tag) and mCherry under the control of the Ptrc-2 promoter; used as a negative control in the sgRNA assay Gibson g-Block + Backbone with UNS 2, 4
reporter mCherry + sgRNA Half of the full construct, to test the function of the sgRNA (ensure it is targetting the correct sequence) Gibson assembly of sgRNA g-Block into reporter mCherry, with UNS 5 extension on backbone 4, 5
reporter mCherry + sgRNA+ AcrIIA4 Incorporation of the Anti-CRISPR protein into the sgRNA construct to have both expressed simultaneously and test the function of the Anti-CRISPR Gibson assemly of AcrIIA4 g-block into reporter mCherry + sgRNA, with UNS 6 extension on backbone 4, 6
reporter construct Composite 3253 Light regulated reporter switch, alternating between mCherry and YFP flourescence Gibson assembly of reporter YFP (LacILOV + YFP) into reporter mCherry with UNS 3 extension on backbone 3, 2
full construct Light-regulated switch controlling the activity of the CRISPR-Cas9 system. Gibson assembly of AcrIIA4 subpart (LacILOV + AcrIIA4 + YFP) into reporter mCherry + sgRNA, with UNS 3 extension on bacbone 3, 6
Figure 1.1: Other parts

References

  1. Canadian Biosafety Standards and Guidelines - https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html
  2. Biosafety Team in the Office of Environmental Health and Safety - https://ehs.utoronto.ca/our-services/biosafety/
  3. NIH Guidelines - https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html
  4. iGEM Risk Groups - https://2017.igem.org/Safety/Risk_Groups

General Pages

Wet Lab

Dry Lab

Human Practices

iGEM Toronto