Difference between revisions of "Team:Waterloo/Description"

Line 1: Line 1:
−
{{Waterloo}}
+
{{Template:Waterloo}}
−
<html>
+
<!-- Requirements Page -->
 +
<html lang="en" ng-app="waterloo">
  
 +
<head>
 +
  <meta charset="utf-8">
 +
  <title>Waterloo iGEM</title>
 +
</head>
  
 +
<body>
 +
  <div class="header" ng-include="'../HomePage/Waterloo_Header.html'"></div>
  
−
<div class="column full_size">
+
  <!-- Just so you know, the requirements-text and elaboration-text classes are supposed to be stored at 2016.igem.org/Team:Waterloo/style.css
 +
    but for some reason identically named classes seem to be getting loaded from somewhere else? Cody pls help. -->
  
−
<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
+
  <div class="wcontent-box">
 +
    <div class="wcontent-title">
 +
      Motivation
 +
    </div>
 +
    <div class="wcontent">
 +
      <p>
 +
        Human neurodegenerative diseases (NDDs) like Creutzfeldt-Jakob, Alzheimer’s, and Parkinson’s disease are associated with multiple metabolic complications in neurons. To name a known few, the metabolism of cholesterol, copper, iron, heme, NAD+, and some
 +
        neurotransmitters have all been shown to be affected by prion proteins affiliated with these NDDs1,2,3,4,5. It is important to continue studying the metabolic implications of NDDs to further elucidate their pathological mechanisms. For diseases
 +
        like Alzheimer’s, years of work have been put in, yet treatment and therapy are only beginning to progress more rapidly. We hope to contribute to this field by providing a synthetic biology approach that enables an alternative method for metabolic
 +
        studies in neurons affected by prion proteins.
 +
      </p>
 +
      <p>
 +
        This year, we’ve designed a negative feedback loop using a novel regulatory element capable of overexpressing or repressing target proteins upon transitioning from a neutral state to a prion state. As a proof-of-concept, we use the Sup35 prion protein
 +
        associated with the [psi-] (healthy) / [PSI+] (disease-like) state of Saccharomyces cerevisiae cells. We use read-through of a premature stop codon in fusions with Hsp104 or dCas9 to respectively overexpress or repress Hsp104. Both overexpression
 +
        and repression of Hsp104, through different mechanisms outlined here, cures the [PSI+] state – from priON to OFF.
 +
      </p>
 +
      <p>
 +
        This system’s design consists of a three-step process: trigger, signal, and control.
 +
      </p>
 +
    </div>
 +
  </div>
  
 +
  <div class="wcontent-box">
 +
    <div class="wcontent-title">
 +
      Trigger
 +
    </div>
 +
    <div class="wcontent">
 +
      <p>
 +
        Sup35’s translational terminator activity, which allows it to remove ribosomes from mRNAs when they’ve reached a stop codon, is lost during the [PSI+] state which we induce via introduction of an extra Sup35 gene fused to a green fluorescent protein (GFP)
 +
        on a plasmid. Our specially designed fusion of cyan fluorescent protein (CFP) and a Sup35 disaggregase (Hsp104) has a premature stop codon positioned upstream of CFP’s fluorophore. The loss in translation terminator activity upon transitioning
 +
        to [PSI+] increases the likelihood of ribosomes translating the entire CFP::Hsp104 fusion protein due to read-through of the premature stop codon. This essentially triggers the increased production of CFP::Hsp104 in response to developing [PSI+]state.
 +
      </p>
 +
      <p>
 +
        By inputting dCas9 in lieu of the Hsp104 ORF in the plasmid, we can use mathematical modeling to predict the ability of optimized sgRNAs to repress the expression of the native Hsp104 gene in the S. cerevisiae genome.. This is based on the concept that
 +
        overexpression of Hsp104 beyond an upper threshold cures the [PSI+] state, and repression below a lower threshold prevents propagation of the Sup35 aggregates to daughter cells, thus curing future generations.
 +
      </p>
 +
    </div>
 +
  </div>
  
−
<h5>What should this page contain?</h5>
+
  <div class="wcontent-box">
−
<ul>
+
    <div class="wcontent-title">
−
<li> A clear and concise description of your project.</li>
+
      Signal
−
<li>A detailed explanation of why your team chose to work on this particular project.</li>
+
    </div>
−
<li>References and sources to document your research.</li>
+
    <div class="wcontent">
−
<li>Use illustrations and other visual resources to explain your project.</li>
+
      <p>
−
</ul>
+
        While the [PSI+] state develops, the cell’s GFP signal increases due to Sup35::GFP overexpression. In response, the CFP signal also increases as it is triggered by the loss of translational terminator activity due to aggregated Sup35. With fluorimetry,
−
 
+
        we monitor the changes in the GFP and CFP signal shown in our Results Section. We also observe the changes between [psi-] and [PSI+] through other phenotypic outputs: the colour of the colonies, and the cellular granularity determined by flow
−
 
+
        cytometry.
−
</div>
+
      </p>
−
 
+
    </div>
−
<div class="column full_size" >
+
−
 
+
−
<h5>Advice on writing your Project Description</h5>
+
−
 
+
−
<p>
+
−
We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.  
+
−
</p>
+
−
 
+
−
<p>
+
−
Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column half_size" >
+
−
 
+
−
<h5>References</h5>
+
−
<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column half_size" >
+
−
<h5>Inspiration</h5>
+
−
<p>See how other teams have described and presented their projects: </p>
+
−
 
+
−
<ul>
+
−
<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
+
−
<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
+
−
<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
+
−
</ul>
+
−
</div>
+
−
 
+
  
 +
</body>
  
 
</html>
 
</html>

Revision as of 15:50, 15 October 2016

Waterloo iGEM
Motivation

Human neurodegenerative diseases (NDDs) like Creutzfeldt-Jakob, Alzheimer’s, and Parkinson’s disease are associated with multiple metabolic complications in neurons. To name a known few, the metabolism of cholesterol, copper, iron, heme, NAD+, and some neurotransmitters have all been shown to be affected by prion proteins affiliated with these NDDs1,2,3,4,5. It is important to continue studying the metabolic implications of NDDs to further elucidate their pathological mechanisms. For diseases like Alzheimer’s, years of work have been put in, yet treatment and therapy are only beginning to progress more rapidly. We hope to contribute to this field by providing a synthetic biology approach that enables an alternative method for metabolic studies in neurons affected by prion proteins.

This year, we’ve designed a negative feedback loop using a novel regulatory element capable of overexpressing or repressing target proteins upon transitioning from a neutral state to a prion state. As a proof-of-concept, we use the Sup35 prion protein associated with the [psi-] (healthy) / [PSI+] (disease-like) state of Saccharomyces cerevisiae cells. We use read-through of a premature stop codon in fusions with Hsp104 or dCas9 to respectively overexpress or repress Hsp104. Both overexpression and repression of Hsp104, through different mechanisms outlined here, cures the [PSI+] state – from priON to OFF.

This system’s design consists of a three-step process: trigger, signal, and control.

Trigger

Sup35’s translational terminator activity, which allows it to remove ribosomes from mRNAs when they’ve reached a stop codon, is lost during the [PSI+] state which we induce via introduction of an extra Sup35 gene fused to a green fluorescent protein (GFP) on a plasmid. Our specially designed fusion of cyan fluorescent protein (CFP) and a Sup35 disaggregase (Hsp104) has a premature stop codon positioned upstream of CFP’s fluorophore. The loss in translation terminator activity upon transitioning to [PSI+] increases the likelihood of ribosomes translating the entire CFP::Hsp104 fusion protein due to read-through of the premature stop codon. This essentially triggers the increased production of CFP::Hsp104 in response to developing [PSI+]state.

By inputting dCas9 in lieu of the Hsp104 ORF in the plasmid, we can use mathematical modeling to predict the ability of optimized sgRNAs to repress the expression of the native Hsp104 gene in the S. cerevisiae genome.. This is based on the concept that overexpression of Hsp104 beyond an upper threshold cures the [PSI+] state, and repression below a lower threshold prevents propagation of the Sup35 aggregates to daughter cells, thus curing future generations.

Signal

While the [PSI+] state develops, the cell’s GFP signal increases due to Sup35::GFP overexpression. In response, the CFP signal also increases as it is triggered by the loss of translational terminator activity due to aggregated Sup35. With fluorimetry, we monitor the changes in the GFP and CFP signal shown in our Results Section. We also observe the changes between [psi-] and [PSI+] through other phenotypic outputs: the colour of the colonies, and the cellular granularity determined by flow cytometry.