Team:MIT/Results

From 2012.igem.org

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{{MIT-header2}}
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DEPRECATED.  DO NOT EDIT.
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{{MIT-style-team}}
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<html>
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  <link href="http://ajax.googleapis.com/ajax/libs/jqueryui/1.8/themes/base/jquery-ui.css" rel="stylesheet" type="text/css"/>
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  <script src="http://ajax.googleapis.com/ajax/libs/jqueryui/1.8/jquery-ui.min.js"></script>
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</head>
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<body>
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<div id="col_nav">
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    <div id="accordion">
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<h3><a href="#">Overview</a></h3>
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<div class="col_list">
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<ul>
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    <li id="aa1">Placeholder</li>
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</ul>
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    </div><!-- end overview -->
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<h3><a href="#">Biobricks</a></h3>
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</ul>
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<h3><a href="#">Foundational</a></h3>
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<div class="col_list">
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<ul>
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    <li id="b1">In Vitro </li>
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    <li id="b2">Nucleic Acid Delivery</li>
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            <li id="b3">In Vivo Strand Displacement </li>
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</ul>
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    </div><!-- end foundational -->
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<h3><a href="#">Sensing</a></h3>
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            <li id="iv1">Modeling</li>
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            <li id="iv2">In Vitro Sensing</li>
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          </ul>
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        </div><!-- end sensing-->
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<h3><a href="#">Processing</a></h3>
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          <ul>
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            <li id="m1"> Modeling </li>
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            <li id="m2"> In Vitro Not Gate </li>
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            <li id="m3"> Hammerhead Ribozymes </li>
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          </ul>
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        </div><!-- end processing-->
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<h3><a href="#">Actuation</a></h3>
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          <ul>
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            <li id="m4"> Modeling </li>
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            <li id="m5"> In Vitro  </li>
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            <li id="m6"> In Vivo </li>
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<div class="bio" id="b1bio">
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<b> RNA Strand Displacement In Vitro </b>
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<br>
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<br>
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<b> Previously:
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<br>
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<br> In 2011, Lulu Qian and Erik Winfree of Caltech, published a paper "Scaling Up Digital Circuit Computation with DNA Strand Displacement cascades. This paper demonstrated how scalable DNA strand displacement based logic circuits are capable of processing functions as difficult as square roots.
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<br>
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<br>
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<b> MIT iGEM 2012: </b>
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<br> Before our team attempted to bring the mechanism of strand displacement into an inv vivo context, we first decided to try assaying strand displacement in vitro using RNA. We used 2'-O-Methyl RNA strands that have never been proved before to strand displace in vitro. Before creating our own constructs, we decided to use sequences from Qian/Winfree and adapt them to RNA.
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<br>
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<b> Foundational Experiment: </b>
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<br>
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Figure A shows a foundational experiment with RNA strand displacement in vitro, performed on a plate reader. The negative control, in black, is a well that receives just an anneal reporter complex. This reporter has gate strand T*S6* that is tagged with a fluorophore, 3' ROX. The top strand of the complex, the output strand, S6 which is complementary to S6*, is tagged with a quencher, that absorbs fluorescence from the fluorophore. When the two strands of the reporter are annealed, no fluorescence should be observed.  The positive control, in red, is the input strand, TS6, annealed to the gate strand, T*S6* tagged with ROX. This is what we would expect the product of a strand displacement reaction to behave like. We can see that in the experimental well when the input is present, it can bind to the exposed T* domain of the reporter and displace the output, S6-quencher, strand yielding a fluorescent complex and waste.
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<br>
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<b> Figure A </b>
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<img src="https://static.igem.org/mediawiki/2012/e/ea/Foundation1MIT.png"/>
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</div>
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<div class="bio" id="b2bio">
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<b> Nucleic Acid Delivery </b>
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<b> Delivery of Plasmid DNA to Mammalian Cells </b>
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<br> Constitutive Expression of fluorescent markers etc
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<b> Delivery of 2'-O-Me RNA to Mammalian Cells </b>
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<br>
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<br> Movie of HEK293 cell being transfected with T*-S6*-ROX, as well as static images
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<br> 15,20,25,30 pmol ratio data from FACS
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<b> Inducible Control of Protein Expression </b>
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<br> DOX curve
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<b> Delivery of Plasmid DNA which transcribes short RNA Inputs </b>
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<br> FF1 Knockdown Data
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</div>
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<div class="bio" id="b3bio">
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<b>In Vivo RNA Strand Displacement </b>
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<br>
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<b> Strategy 1: Lipofectamine 2000 Transfection of RNA version of Reporter from Winfree/QIan 2011 Paper</b>
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<br> Images will go here from April 24th experiment  - display red fluorescence in all wells, including those that only got reporter or the wrong input - also see red vesicles indicating reporter comes apart inside the vesicles
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<br>
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<br>
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<br> Strategy 2: Switch Transfection reagent to RNAiMAX </b>
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<br> RNAiMAX is supposed to be a better transfection reagent for double stranded RNA
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<br> Images will go here from experiment from June 13th onward where we do not see red vesicles, however we still see whole cell red fluorescence
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<b> Strategy 3: Tag RNA strand with an Alexa Fluorophore to act as a transfection marker </b>
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<b> Strategy 4: Create DNA plasmids driving transcription of RNA inputs, while transfecting RNA Reporter </b>
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<b> Strategy 4: Nucleofect RNA reporter, RNA inputs </b>
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<b> [Strategy 5]: Redesign RNA Reporter </b>
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</div>
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</html>
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Latest revision as of 16:41, 1 October 2012

DEPRECATED. DO NOT EDIT.

Retrieved from "http://2012.igem.org/Team:MIT/Results"