Difference between revisions of "Team:NTU-Singapore/Demonstrate"

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{{NTU-Singapore}}
 
{{NTU-Singapore}}
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<html>
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<html lang="en">
  
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<head>
  
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<div class="column full_size" >
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    <meta charset="utf-8">
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    <meta http-equiv="X-UA-Compatible" content="IE=edge">
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    <meta name="viewport" content="width=device-width, initial-scale=1">
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    <meta name="description" content="">
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    <meta name="author" content="">
  
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<p>Here you can describe the results of your project and your future plans. </p>
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    <title>NTU-Singapore</title>
  
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<h5>What should this page contain?</h5>
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    <!-- Fonts -->
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<ul>
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        <script src="https://use.fontawesome.com/597ba5ca72.js"></script>
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<li> Clearly and objectively describe the results of your work.</li>
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<link href="https://fonts.googleapis.com/css?family=Raleway" rel="stylesheet">
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<li> Future plans for the project </li>
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<link href="https://fonts.googleapis.com/css?family=Amatic+SC:700|Annie+Use+Your+Telescope|Gloria+Hallelujah|Just+Me+Again+Down+Here|Nothing+You+Could+Do|Reenie+Beanie|Rock+Salt|Schoolbell|Shadows+Into+Light|Sue+Ellen+Francisco" rel="stylesheet">
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<li> Considerations for replicating the experiments </li>
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</ul>
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</head>
  
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</div>
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<body>
  
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<div class="column half_size" >
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    <div id="wrapper">
  
 +
        <!-- Navigation -->
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      <nav class="navbar navbar-inverse navbar-fixed-top" role="navigation">
 +
            <!-- Brand and toggle get grouped for better mobile display -->
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            <div class="navbar-header">
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                <button type="button" class="navbar-toggle" data-toggle="collapse" data-target=".navbar-ex1-collapse">
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                    <span class="sr-only">Toggle navigation</span>
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                    <span class="icon-bar"></span>
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                    <span class="icon-bar"></span>
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                    <span class="icon-bar"></span>
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                </button>
 +
               
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            </div>
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            <!-- Top Menu Items -->
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            <ul class="nav navbar-right top-nav" style="margin: auto; width: 1063px;">
  
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<h5> Project Achievements </h5>
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                <li class="dropdown" style="margin-left:0px;">
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<a href="https://2016.igem.org/Team:NTU-Singapore" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x fa-home"></i> <span class="navbar-name"> NTU-SG</span> </a>
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</li>
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                <li class="dropdown">
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                    <a href="https://2016.igem.org/Team:NTU-Singapore/Collaborations" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x fa-globe"></i><span class="navbar-name"> Collaboration</span></a>
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</li>
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<li class="dropdown">
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<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x fa-flask"></i><span class="navbar-name"> Projects</span><b class="caret"></b></a>
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<div class="drop-menu">
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<ul class="dropdown-menu dropdown-menu-project" style="margin: 0px;  left: -40px;">
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Interlab"><i class="fa fa-2x fa-balance-scale"></i><span style="top: -34px; float: left; left: 50px;"class="navbar-name"> Interlab Measurement </span> </a>
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</li>
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Description"  style="top: -20px; position: relative;"><i class="fa fa-fw fa-2x fa-book"></i><span style="left: 9px;" class="navbar-name"> Description</span> </a>
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</li>
  
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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>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Proof" style="height: 49px;"><i class="fa fa-2x  fa-bar-chart" style="float: left;"></i><span style="width: 96px;
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left: 10px;
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float: left;
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margin-left: 6px;
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top: -6px;" class="navbar-name"> Proof of concept </span></a>
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</li>
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Demonstrate"><i class="fa fa-2x  fa-space-shuttle"></i><span style="left: 9px;" class="navbar-name"> Demonstrate</span> </a>
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</li>
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</ul>
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</div>
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</li>
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 +
<li class="dropdown">
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<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x fa-gears"></i><span class="navbar-name"> Parts</span>  <b class="caret"></b></a>
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<div class="drop-menu">
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<ul class="dropdown-menu" style="margin: 0px; left: -42px;">
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Basic_Part"><i class="fa fa-fw fa-2x fa-gear"></i><span style="left: 8px;"class="navbar-name"> Basic Parts</span> </a>
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</li>
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<li>
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<a style="height: 49px" href="https://2016.igem.org/Team:NTU-Singapore/Composite_Part"><i style="float: left;" class="fa fa-fw fa-2x fa-briefcase"></i><span style="    float: left;
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width: 96px;
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top: -6px;
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left: 13px;" class="navbar-name"> CRISPR<span style="text-transform: lowercase;">y</span> Collection</span> </a>
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</li>
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 +
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</ul>
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</div>
 +
 +
</li>
 +
 +
 +
<li class="dropdown">
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<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x fa-key"></i><span class="navbar-name"> Team</span>  <b class="caret"></b></a>
 +
<div class="drop-menu">
 +
<ul class="dropdown-menu" style="margin: 0px; left: -50px;">
 +
<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Team"><i class="fa fa-2x fa-flag-checkered" aria-hidden="true"></i><span style="left: 13px;"class="navbar-name">Adventurers</span> </a>
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</li>
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/Attributions"><i class="fa fa-2x fa-key" aria-hidden="true"></i><span style="left: 13px;" class="navbar-name">Attribution</span> </a>
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</li>
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<li>
 +
<a style="height: 49px" href="https://2016.igem.org/Team:NTU-Singapore/Notebook"><i class="fa fa-2x fa-file-text-o" aria-hidden="true"></i>
 +
<span style=" left: 13px;" class="navbar-name">Notebook</span> </a>
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</li>
 +
 +
 +
</ul>
 +
</div>
  
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<ul>
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</li>
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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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</ul>
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</div>
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                                      <li class="dropdown">
 +
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="fa fa-2x  fa-group"></i><span class="navbar-name"> Human Practice</span>  <b class="caret"></b></a>
 +
<div class="drop-menu">
 +
<ul class="dropdown-menu dropdown-menu-humanprac" style="margin: 0px">
 +
<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/HP/Silver"><i class="fa fa-fw fa-2x fa-user"></i><span class="navbar-name"> Silver</span> </a>
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</li>
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<li>
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<a href="https://2016.igem.org/Team:NTU-Singapore/HP/Gold"><i class="fa fa-fw fa-2x fa-envelope"></i><span class="navbar-name"> Gold</span> </a>
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</li>
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</ul>
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</div>
 +
</li>
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 +
 
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            </ul>
 +
                </nav>
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 +
                <div class="jumbotron content-page" style="font-family: 'Just Me Again Down Here', cursive; padding-top:150px; height: 340px;">
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<h1 style="font-size: 70px;"><span style="color: #0089A7; ">CRISPRy</span>! I choose you!</h1>
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<p style="font-family: 'Annie Use Your Telescope', cursive; font-size: 31px;">To infinity! And beyond!!</p>
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</div>
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 +
<div class="page-header">
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<h2>Project Truncation</h2>
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</div>
 +
<div class="well">
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<p>Knowing that several of our truncated mutants is able to activate our reporter gene, we now turned our attention to using dCas9 for the activation of an endogenous gene. The difference between reporter gene and endogenous gene activation is the copy number of our template within the cell, with the former in larger amounts than the latter.</p>
 +
 
 +
<p>We tried to activate an endogenous gene, ADAR2 in HEK293 cells. ADAR2 is a dsRNA-specific adenosine deaminase involved in A-I conversion. Such conversions would result in amino-acid substitutions, splice junction alterations, RNA stability chnages, etc. It has been shown in human patient, downregulation of ADAR2 expression and hence underediting Glu2 has lead to <a href="https://www.ncbi.nlm.nih.gov/pubmed/15624111" style="color: cadetblue; font-weight: bold;">amyotrophic lateral sclerosis diseases</a>. GluR2 are generated postransciptionally by RNA editing. Unedited GluR2 have high Ca2+ permeability. This high Ca2+ flux into cell has caused neuronal death in mice model. Thus, activating ADAR2 with dCas9 might be one of the potential future treatment for amyotrophic lateral sclerosis diseases.</p>
 +
 
 +
<p> Our advisor, Dr. Yuan Ming, is also using dCas9-VPR for the induction of ADAR3 and ADAR2 expression in H1 human embryonic stem cells. We used the gRNA designed by Dr. Yuan Ming to test out if our truncated dCas9 could activate endogenous ADAR2 and ADAR3 in HEK293 and H1 cells. After 48 hours of transfection, RNA is extracted from the cells and used for RT-qPCR analysis. GAPDH RNA is used as control for normalisation.</p>
 +
 
 +
<div style="margin: auto; width: 680px;">
 +
 
 +
<img class="content-img" src="https://static.igem.org/mediawiki/2016/2/2a/T--NTU-Singapore--ADAR2.png" alt="" style="width:680px; height:400px; box-shadow: none;">
 +
 +
<p style="font-size:18px; text-align: center;">ADAR2 activation in HEK293 cells with only one gRNA used.</p>
 +
    </div>
 +
 +
<p>ADAR2 activation with only one gRNA seems to correlate with our proof of concept experiment. ∆REC1-3 and ∆HNH mutants are able to retain ~80% WT activity while ∆RuvCIII-2 ∆HNH double truncation mutant still has >50% activity. We then tried out these truncated mutants for ADAR3 activation on H1 embryonic stem cells cultured by Dr. Yuan Ming. ADAR3 is also a dsRNA-specific adenosine deaminase involved in A-I conversion. However, ADAR3 specifically expressed in neuronal cells. ADAR3 activation predisposed human ES cells to a neuronal fate.</p>
 +
 
 +
<div style="margin: auto; width: 680px;">
 +
 
 +
<img class="content-img" src="https://static.igem.org/mediawiki/2016/3/37/T--NTU-Singapore--ADAR3.png" alt="" style="width:680px; height:400px; box-shadow: none;">
 +
 +
<p style="font-size:18px; text-align: center;">ADAR3 activation in H1 embryonic stem cells with only one gRNA used.</p>
 +
    </div>
  
 +
<p>Only ∆REC1-3 truncated mutant is able to activate ADAR3 in H1 cells. However, further testing have to be done on other truncations to determine parameters for optimal performance of these truncations.</p>
  
−
<div class="column half_size" >
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</div>
  
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<h5>Inspiration</h5>
 
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<p>See how other teams presented their results.</p>
 
−
<ul>
 
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
 
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
 
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
 
−
</ul>
 
  
 +
 +
 +
<div style="height: 150px; background-color: rgba(120,194,196,1);">
 +
 
</div>
 
</div>
 +
        </div>
 +
        <!-- /#page-wrapper -->
  
 +
    </div>
 +
    <!-- /#wrapper -->
  
 +
</body>
  
 
</html>
 
</html>

Latest revision as of 03:32, 20 October 2016

NTU-Singapore

CRISPRy! I choose you!

To infinity! And beyond!!

Project Truncation

Knowing that several of our truncated mutants is able to activate our reporter gene, we now turned our attention to using dCas9 for the activation of an endogenous gene. The difference between reporter gene and endogenous gene activation is the copy number of our template within the cell, with the former in larger amounts than the latter.

We tried to activate an endogenous gene, ADAR2 in HEK293 cells. ADAR2 is a dsRNA-specific adenosine deaminase involved in A-I conversion. Such conversions would result in amino-acid substitutions, splice junction alterations, RNA stability chnages, etc. It has been shown in human patient, downregulation of ADAR2 expression and hence underediting Glu2 has lead to amyotrophic lateral sclerosis diseases. GluR2 are generated postransciptionally by RNA editing. Unedited GluR2 have high Ca2+ permeability. This high Ca2+ flux into cell has caused neuronal death in mice model. Thus, activating ADAR2 with dCas9 might be one of the potential future treatment for amyotrophic lateral sclerosis diseases.

Our advisor, Dr. Yuan Ming, is also using dCas9-VPR for the induction of ADAR3 and ADAR2 expression in H1 human embryonic stem cells. We used the gRNA designed by Dr. Yuan Ming to test out if our truncated dCas9 could activate endogenous ADAR2 and ADAR3 in HEK293 and H1 cells. After 48 hours of transfection, RNA is extracted from the cells and used for RT-qPCR analysis. GAPDH RNA is used as control for normalisation.

ADAR2 activation in HEK293 cells with only one gRNA used.

ADAR2 activation with only one gRNA seems to correlate with our proof of concept experiment. ∆REC1-3 and ∆HNH mutants are able to retain ~80% WT activity while ∆RuvCIII-2 ∆HNH double truncation mutant still has >50% activity. We then tried out these truncated mutants for ADAR3 activation on H1 embryonic stem cells cultured by Dr. Yuan Ming. ADAR3 is also a dsRNA-specific adenosine deaminase involved in A-I conversion. However, ADAR3 specifically expressed in neuronal cells. ADAR3 activation predisposed human ES cells to a neuronal fate.

ADAR3 activation in H1 embryonic stem cells with only one gRNA used.

Only ∆REC1-3 truncated mutant is able to activate ADAR3 in H1 cells. However, further testing have to be done on other truncations to determine parameters for optimal performance of these truncations.