Difference between revisions of "Team:New York City/Design"

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{{New_York_City}}
 
{{New_York_City}}
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<html>
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<html lang="en">
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<head><style>
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<div class="column full_size">
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        .container {
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<h1>Design</h1>
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            align-items: center;
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<p>
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            max-width: 960px;
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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        }
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</p>
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        a{
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            color:#7B84ED;
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<p>
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2018.igem.org/Team:New_York_City/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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        p{
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</p>
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        }
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        .shiny{
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            color:#7B84ED;
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        }
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        .headimage{
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            border-radius: 15px;
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        }
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.social{
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            max-width: 50px;
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            max-height: auto;
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        }
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img { max-width: 40%;}
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    </style>
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</head>
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<body>
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    <div class="container">
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        <nav class="sticky-top py-3 navbar navbar-expand-lg navbar-light mr-auto">
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            <div class="container d-flex flex-column flex-md-row justify-content-between">
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                <div class="navbar-brand"><a href="https://2018.igem.org/Team:New_York_City">New York City iGEM</a></div>
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                <div class="collapse navbar-collapse" id="navbarSupportedContent">
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                    <ul class="navbar-nav mr-auto">
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                        <li class="nav-item dropdown">
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                            <a class="nav-link dropdown-toggle py-2 d-none d-md-inline-block" href="#" id="navbarPR"
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                                role="button" data-toggle="dropdown" aria-haspopup="true" aria-expanded="false">
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                                Project
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                            </a>
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                            <div class="dropdown-menu" aria-labelledby="navbarPR">
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/HD">What is
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                                    Huntington's?</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Description">Description</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Design">Design</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Model">Model</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/InterLab">InterLab</a>
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                            </div>
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                        </li>
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                        <li class="nav-item dropdown">
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                            <a class="nav-link dropdown-toggle py-2 d-none d-md-inline-block" href="#" id="navbarNB"
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                                role="button" data-toggle="dropdown" aria-haspopup="true" aria-expanded="false">
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                                Notebook
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                            </a>
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                            <div class="dropdown-menu" aria-labelledby="navbarNB">
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Notebook">Notebook</a>
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                            </div>
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                        </li>
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                        <li class="nav-item dropdown">
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                            <a class="nav-link dropdown-toggle py-2 d-none d-md-inline-block" href="#" id="navbarHP"
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                                role="button" data-toggle="dropdown" aria-haspopup="true" aria-expanded="false">
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                                Human Practices
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                            </a>
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                            <div class="dropdown-menu" aria-labelledby="navbarHP">
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Human_Practices">Human
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                                    Practices</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Integrated_Practices">Integrated
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                                    Practices</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Public_Engagement">Public
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                                    Engagement</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Collaborations">Collaborations</a>
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                            </div>
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                        </li>
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                        <li class="nav-item dropdown">
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                            <a class="nav-link dropdown-toggle py-2 d-none d-md-inline-block" href="#" id="navbarT"
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                                role="button" data-toggle="dropdown" aria-haspopup="true" aria-expanded="false">
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                                Team
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                            </a>
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                            <div class="dropdown-menu" aria-labelledby="navbarT">
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Team">Team</a>
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                                <a class="dropdown-item" href="https://2018.igem.org/Team:New_York_City/Attributions">Attributions</a>
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                            </div>
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                        </li>
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                    </ul>
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                </div>
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            </div>
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        </nav>
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    </div>
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<div class="container py-0 align-middle">
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    <h2 align="center">Project Design</h2>
 
</div>
 
</div>
  
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<div class="container">
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    <p class="lead">Our goal is to create a cure for Huntington’s Disease (HD) by developing a RNA strand displacement technology that can target and block mutated huntingtin (HTT) mRNA strands and replace them with corrected strands for proper protein synthesis. Since an increased number of CAG repeats in the HTT gene coding for the polyglutamine tail characteristic of HD contributes to neurotoxicity in this disease, we decided to find a cure for HD in the level of RNA. Last year, we designed a modified plasmid consisting of a chaperone strand and a corrected HTT mRNA strand using the software programs Vienna and mFold. The chaperone strand contains a small RNAi like toehold sequence designed to bind to the hairpin loop sequence of mutated HTT mRNA strands. Upon binding of the chaperone strand to the mutated mRNA strand, the corrected HTT mRNA strand would be released into the cytoplasm for proper protein synthesis.</p>
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<div class="column two_thirds_size">
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<h3>What should this page contain?</h3>
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<ul>
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<li>Explanation of the engineering principles your team used in your design</li>
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<li>Discussion of the design iterations your team went through</li>
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<li>Experimental plan to test your designs</li>
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</ul>
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</div>
 
</div>
  
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<div class="column third_size">
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<div class="container">
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<div class="highlight decoration_A_full">
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    <p class="lead">After developing this modified plasmid, we looked into in vitro models to test the efficacy of our RNA strand displacement technology. Our literature review into research involving HD cell lines revealed that not much is known about the role of normal, endogenous HTT protein in non-neuronal cells. Therefore, we decided to initially test the efficacy of our modified plasmid in HeLa cell lines modified to express a polyglutamine tail adhered to cyan fluorescent protein (CFP). After maintaining the HeLa/polyQ-mCFP cell line, we transfected the cells at two different concentrations, performed cell lysis, followed by western blotting. Since the mutated HTT protein would be too large to blot, we used a probe for CFP as a proxy for the quantity of HTT protein in cells. The western blot was used to determine whether transfecting cells at increasing concentrations of the modified plasmid would result in a decreased quantities of HTT protein.</p>
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<h3>Inspiration</h3>
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<ul>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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</ul>
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</div>
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</div>
 
</div>
  
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<div align="center">
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            <img src="https://static.igem.org/mediawiki/2018/1/18/T--New_York_City--des1.jpg"/>
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            </div>
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<div align="center">
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            <img src="https://static.igem.org/mediawiki/2018/4/41/T--New_York_City--des2.jpg"/>
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            </div>
  
  
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<br/>    <div class="container" align="center">
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        <div class="row" align="center">
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            <div class="col-sm-3">
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            </div>
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            <div class="col-sm-2">
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                <a href="https://www.facebook.com/HDResolution/"><img class="social" src="https://static.igem.org/mediawiki/2018/6/61/T--New_York_City--fb.png"></a>
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            </div>
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            <div class="col-sm-2">
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                <a href="https://twitter.com/HDNYC_iGEM"><img class="social" src="https://static.igem.org/mediawiki/2018/4/46/T--New_York_City--twit.png"></a>
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            </div>
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            <div class="col-sm-2">
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                <a href="https://www.instagram.com/nyc_igem/"><img class="social" src="https://static.igem.org/mediawiki/2018/0/0a/T--New_York_City--ig.png"></a>
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            </div>
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            <div class="col-sm-3">
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            </div>
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        </div>
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        <br />
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    </div>
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</body>
 
</html>
 
</html>

Latest revision as of 01:38, 17 October 2018

Project Design

Our goal is to create a cure for Huntington’s Disease (HD) by developing a RNA strand displacement technology that can target and block mutated huntingtin (HTT) mRNA strands and replace them with corrected strands for proper protein synthesis. Since an increased number of CAG repeats in the HTT gene coding for the polyglutamine tail characteristic of HD contributes to neurotoxicity in this disease, we decided to find a cure for HD in the level of RNA. Last year, we designed a modified plasmid consisting of a chaperone strand and a corrected HTT mRNA strand using the software programs Vienna and mFold. The chaperone strand contains a small RNAi like toehold sequence designed to bind to the hairpin loop sequence of mutated HTT mRNA strands. Upon binding of the chaperone strand to the mutated mRNA strand, the corrected HTT mRNA strand would be released into the cytoplasm for proper protein synthesis.

After developing this modified plasmid, we looked into in vitro models to test the efficacy of our RNA strand displacement technology. Our literature review into research involving HD cell lines revealed that not much is known about the role of normal, endogenous HTT protein in non-neuronal cells. Therefore, we decided to initially test the efficacy of our modified plasmid in HeLa cell lines modified to express a polyglutamine tail adhered to cyan fluorescent protein (CFP). After maintaining the HeLa/polyQ-mCFP cell line, we transfected the cells at two different concentrations, performed cell lysis, followed by western blotting. Since the mutated HTT protein would be too large to blot, we used a probe for CFP as a proxy for the quantity of HTT protein in cells. The western blot was used to determine whether transfecting cells at increasing concentrations of the modified plasmid would result in a decreased quantities of HTT protein.