Difference between revisions of "Team:BGIC-Union"

 
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<html>
 
<html>
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     <meta charset="utf-8">
 
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     <title>Team：BGIC-Union/PROJECT</title>
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     <title>Team：BGIC-Union</title>
  
 
   <head>
 
   <head>
 
    
 
    
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        <link href='http://fonts.googleapis.com/css?family=Lato:400,300' rel='stylesheet' type='text/css'>
 
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        <link href='http://fonts.googleapis.com/css?family=Raleway:400,300,500' rel='stylesheet' type='text/css'>
 
  
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  <!-- Bootstrap core CSS -->
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    <link href="https://2017.igem.org/Template:BGIC-Union/bootstrap-css?action=raw&amp;ctype=text/css" rel="stylesheet">
  
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<!-- Bootstrap core CSS -->
 
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    <link href="https://2017.igem.org/Template:BGIC-Union/bootstrap-css?action=raw&amp;ctype=text/css" rel="stylesheet">
 
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<link href="https://2017.igem.org/Template:BGIC-Union/animate-css?action=raw&amp;ctype=text/css" rel="stylesheet">
 
 
   <!-- carousel -->
 
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   <link href="https://2017.igem.org/Template:BGIC-Union/owlcarousel-css?action=raw&amp;ctype=text/css" rel="stylesheet">
 
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   <link href=" https://2017.igem.org/Template:BGIC-Union/fontawesomemin2-css?action=raw&amp;ctype=text/css" rel="stylesheet">
  
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   <!-- main -->
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  <script src="https://2017.igem.org/Template:BGIC-Union/hovernew-js?action=raw&amp;ctype=text/javascript"></script>
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  <script src="https://2017.igem.org/Template:BGIC-Union/hoverzoomjs?action=raw&amp;ctype=text/javascript"></script>
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   <style type="text/css">
 
   <style type="text/css">
 
    
 
    
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@import url(http://fonts.googleapis.com/css?family=Montserrat:400,700);
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#top_title,#sideMenu { display: none !important; }
 
#top_title,#sideMenu { display: none !important; }
 
         #content {
 
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#bodyContent
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{
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margin-top：0px !important;
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.mainstyle
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{
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font-family: Comic Sans MS;
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}
 
html, body {
 
html, body {
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     margin: 0 !important;
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     margin: 0 ;
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}
 
body {
 
body {
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   background-color: #ffa07a;
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   font-family: 'Montserrat', sans-serif;
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   margin-keft：150px
 
     font-weight: 400;
 
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     color: #555;
 
     color: #555;
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     -webkit-font-smoothing: antialiased;
 
     -webkit-font-smoothing: antialiased;
 
     -webkit-overflow-scrolling: touch;
 
     -webkit-overflow-scrolling: touch;
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/* Titles */
 
/* Titles */
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h1, h2, h3, h4, h5, h6,p {
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h1, h2, h3, h4, h5, h6, p {
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font-family: 'Montserrat', sans-serif;
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font-family: Comic Sans MS;
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     font-weight: 600;
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     color: #333;
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line-height:30pt;
 
 
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font-size: 35px;
 
font-size: 35px;
 
margin-top: 30px;
 
margin-top: 30px;
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margin-bottom: 50px;
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margin-bottom: 30px;
 
}
 
}
  
  
  
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/* Paragraph  Typographic */
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p {
 
p {
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     line-height: 50px;
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     line-height: 28px;
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     margin-bottom: 35px;
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     margin-bottom: 25px;
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     font-size: 13px;
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font-family: Comic Sans MS;
 
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     color: #20B2AA;
 
     color: #20B2AA;
 
     word-wrap: break-word;
 
     word-wrap: break-word;
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font-size: 18px;
 
     -webkit-transition: color 0.1s ease-in, background 0.1s ease-in;
 
     -webkit-transition: color 0.1s ease-in, background 0.1s ease-in;
 
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     -moz-transition: color 0.1s ease-in, background 0.1s ease-in;
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}
 
}
  
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.copyrights{
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height:0;
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.navbar {
 
.navbar {
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.navbar-inverse {
 
.navbar-inverse {
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}
 
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.navbar-inverse .navbar-nav > li > a {
 
.navbar-inverse .navbar-nav > li > a {
 
color: white;
 
color: white;
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}
 
}
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.navbar-inverse .navbar-nav > li > a:hover {
 
.navbar-inverse .navbar-nav > li > a:hover {
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.navbar-brand {
 
.navbar-brand {
 
font-weight: 700;
 
font-weight: 700;
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font-size: 20px;
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font-size: 40px;
 
letter-spacing: 2px;
 
letter-spacing: 2px;
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backgroud: white;
 
}
 
}
  
 
.navbar-inverse .navbar-brand {
 
.navbar-inverse .navbar-brand {
 
color: white;
 
color: white;
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background-color: #FFA07A;
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.navbar-inverse .navbar-toggle {
 
.navbar-inverse .navbar-toggle {
 
border-color: transparent;
 
border-color: transparent;
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font-size: 12px;
 
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padding-top: 50px;
 
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/* +++++ WRAP SECTIONS +++++ */
 
/* +++++ WRAP SECTIONS +++++ */
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/* +++++ Color Wraps / Blog Page +++++ */
 
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/* Blog Date*/
 
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ba {
 
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/* ================== PORTFOLIO IMAGES HOVER EFFECT ================== */
 
/* ================== PORTFOLIO IMAGES HOVER EFFECT ================== */
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}
 
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/* glyphicon Configuration */
 
  
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.glyphicon {
 
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font-size: 40px;
 
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color: #1abc9c;
 
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}
 
 
   </style>
 
   </style>
−
 
 
    
 
    
 +
  <!-- script -->
 +
  <script src="https://code.jquery.com/jquery-1.10.2.min.js"></script>
 +
 
 +
  <script src="https://2017.igem.org/Template:BGIC-Union/newbootstrap-js?action=raw&amp;ctype=text/javascript"></script>
 +
 +
<!-- hover    --> 
 +
  <script src="https://2017.igem.org/Template:BGIC-Union/hovernew-js?action=raw&amp;ctype=text/javascript"></script>
 +
  <script src="https://2017.igem.org/Template:BGIC-Union/hoverzoomjs?action=raw&amp;ctype=text/javascript"></script>
 
   </head>
 
   </head>
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  <body>
 
  <body>
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                     <li><a href="https://2017.igem.org/Team:BGIC-Union/Team">members</a></li>
 
                     <li><a href="https://2017.igem.org/Team:BGIC-Union/Team">members</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Collaborations">collaborations</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Collaborations">collaborations</a></li>
−
<li><a href="https://2017.igem.org/Team:BGIC-Union/Sponsors">sponsors</a></li>
+
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Attributions">attributions</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Attributions">attributions</a></li>
 
                             </ul>
 
                             </ul>
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                             <ul class="dropdown-menu" >
 
                             <ul class="dropdown-menu" >
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Description">description</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Description">description</a></li>
−
<li><a href="https://2017.igem.org/Team:BGIC-Union/Design”></a>design</li>
+
<li><a href="https://2017.igem.org/Team:BGIC-Union/Design">design</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Results">results</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Results">results</a></li>
−
                                <li><a href="https://2017.igem.org/Team:BGIC-Union/Model">model</a></li>
+
                         
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Demonstrate">demonstrate</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Demonstrate">demonstrate</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Improve">improve</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Improve">improve</a></li>
 +
<li><a href="https://2017.igem.org/Team:BGIC-Union/References">references</a></li>
 
                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
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                         <li class="dropdown ">    <a href="https://2017.igem.org/Team:BGIC-Union/Lab" class="dropdown-toggle" data-toggle="dropdown"> <b class="caret"></b></a>
 
                         <li class="dropdown ">    <a href="https://2017.igem.org/Team:BGIC-Union/Lab" class="dropdown-toggle" data-toggle="dropdown"> <b class="caret"></b></a>
 
                             <ul class="dropdown-menu" role="menu">
 
                             <ul class="dropdown-menu" role="menu">
−
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Experiments">experiments</a></li>
+
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Experiments">protocols</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Notebook">notebook</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/Notebook">notebook</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/InterLab">InterLab</a></li></ul>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/InterLab">InterLab</a></li></ul>
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<li><a href="https://2017.igem.org/Team:BGIC-Union/Composite_Parts">composite parts</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Composite_Parts">composite parts</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Parts_Collection">parts collection</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Parts_Collection">parts collection</a></li>
−
<li><a href="https://2017.igem.org/Team:BGIC-Union/Measurement">measurement</a></li>
+
 
  
 
                             </ul>
 
                             </ul>
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                           <li class="dropdown ">  <a href="https://2017.igem.org/Team:BGIC-Union/Practices-Design" class="dropdown-toggle" data-toggle="dropdown"> <b class="caret"></b></a>
 
                           <li class="dropdown ">  <a href="https://2017.igem.org/Team:BGIC-Union/Practices-Design" class="dropdown-toggle" data-toggle="dropdown"> <b class="caret"></b></a>
 
                             <ul class="dropdown-menu" role="menu">
 
                             <ul class="dropdown-menu" role="menu">
−
                                <li><a href="https://2017.igem.org/Team:BGIC-Union/Practices-Design">timeline</a></li>
+
                             
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/HP/Silver">silver</a></li>
 
                                 <li><a href="https://2017.igem.org/Team:BGIC-Union/HP/Silver">silver</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/HP/Gold_Integrated">gold</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/HP/Gold_Integrated">gold</a></li>
−
<li><a href="https://2017.igem.org/Team:BGIC-Union/Gold_Integrated">integrated</a></li>
+
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Engagement">public engagement</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Engagement">public engagement</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Applied_Design">applied design</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Applied_Design">applied design</a></li>
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<li><a href="https://2017.igem.org/Team:BGIC-Union/Entrepreneurship">entrepreneurship</a></li>
 
<li><a href="https://2017.igem.org/Team:BGIC-Union/Entrepreneurship">entrepreneurship</a></li>
 
                             </ul>
 
                             </ul>
 +
                        </li>
 +
<li class="dropdown ">
 +
                            <a href="https://2017.igem.org/Team:BGIC-Union/Model">Model </a>
 
                         </li>
 
                         </li>
 
<li class="dropdown ">
 
<li class="dropdown ">
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     </div>
 
     </div>
 
     </div>
 
     </div>
−
</nav>           <!-- PAGE CONTENT -->
+
</nav>
−
<img src=“https://static.igem.org/mediawiki/2017/a/a8/Ctdnabgic.jpg” height="400px" />
+
−
        <div class="page-content">
+
−
            <div class="container">
+
−
                <section>
+
</div>
−
                    <!-- +++++ Projects Section +++++ -->
+
 +
  <section >
 +
        <div class="carousel slide" id="carousel-example-generic" data-ride="carousel">
 +
            
 +
 
 +
          <div class="carousel-inner">
 +
          <div class="item active">
 +
          <div class="overlay-slide">
 +
          <img src="https://static.igem.org/mediawiki/2017/archive/8/89/20171021063547%21P3%21%21%21.jpg" width="1300px" class="img-responsive">
 +
          </div>
 +
          <div class="carousel-caption">
 +
              <div class="col-md-12 col-xs-12 text-center">
 +
                      <h1>dCasentry</h1>
 +
              <h3 class="animated2">a <b>dCas9-T7</b>coupled DNA sensor </h3>
 +
              <div class="line"></div>
 +
              <p class="animated3"></p>
 +
              </div>
 +
          </div>
 +
          </div>
 +
 
 +
               
 +
        </div>
 +
  </section>
 +
    <!--/ Slider end -->
 +
    <!--/ Slider end -->
 +
 +
<!-- +++++ Welcome Section +++++ -->
 +
<div id="ww">
 +
    <div class="container">
 +
<div class="row">
 +
<div class="col-lg-8 col-lg-offset-2 ">
 +
 +
<div style="height:122px; width:150px; border-radius:50%; overflow:hidden; ">
 +
<img src="https://static.igem.org/mediawiki/2017/d/db/User.png" height="120px" width="150px">
 +
</div>
 +
</div><h1 class="center">Hi, I am dCasentry,</h1>
 +
<div class="row">
 +
<div class="col-md-6">
 +
<div>dCasentry: A guardian against Lung Cancer</br>
 +
Lung cancer is one of the deadliest malignant tumors in the world, with highest death rate in China. The most widely used lung cancer detection method nowadays remains biopsy, which may cause pain infection and bruising. However, the technic of liquid biopsy obviates this problem by detecting various factors including circulating tumor DNA (ctDNA) in the blood. ctDNA is a short strand of DNA released from tumor cells that can be deemed as the sign of cancer. Although traditional liquid biopsy avoids many side effects, it requires laboratory apparatus such as PCR instrument during detection. Also, some other ctDNA detection systems fail to overcome the difficulty of the extremely low concentration of ctDNA in the blood. To solve those problems, we developed a new liquid biopsy method using dCas9 protein with a much simple procedure while increase the signal with various kinds of output. We hope to adopt free-dried paper chip as our vector of detecting system to simplify its operation as well as storage. Finally, the product will be presented as a kit that contains all the items you needed for convenient machine-free detection.
 +
</br>
 +
<i>Please, click <a href="https://2017.igem.org/Team:BGIC-Union/Project">  here </a> and know more about me as well as the product!</i> </div>
 +
</div>
 +
<div class="col-md-6">
 +
<img src="https://static.igem.org/mediawiki/2017/7/79/FigureDemo.jpg" width="500px" /></div></div><!-- /col-lg-8 -->
 +
</div><!-- /row -->
 +
    </div> <!-- /container -->
 +
</div><!-- /ww -->
 +
   
 +
<!-- +++++ Projects Section +++++ -->
 
 
 
<div class="container pt">
 
<div class="container pt">
−
<div class="row mt centered">
+
<div class="row mt ">
 
<div class="col-lg-4">
 
<div class="col-lg-4">
−
<a href="https://2017.igem.org/Team:BGIC-Union/Description"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/6/68/Description_bgic.png" width="400px" height="300px"></img></a>
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/6/64/Bgicbiosafety.png" width="200px" height="150px"></img></a>
−
<h4>Description</h4>
+
<h4><a href="https://2017.igem.org/Team:BGIC-Union/Parts">Best Parts </a></h4>
 
</div>
 
</div>
 
<div class="col-lg-4">
 
<div class="col-lg-4">
−
<a href="https://2017.igem.org/Team:BGIC-Union/Design"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/f/f3/Designbgic.png" width="400px" height="300px"></img></a>
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/2/26/Applieddesignlogo.png" width="200px" height="150px"></img></a>
−
<h4>Design</h4>
+
<h4><a href="https://2017.igem.org/Team:BGIC-Union/Applied_Design">Applied Design</a></h4>
 
</div>
 
</div>
 
<div class="col-lg-4">
 
<div class="col-lg-4">
−
<a href="https://2017.igem.org/Team:BGIC-Union/Results"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/b/b2/Resultsbgic.png" width="400px" height="300px"></img></a>
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/f/f0/Gold_bgic.png" width="200px" height="150px"></img></a>
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<h4>Results</h4>
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<h4><a href="https://2017.igem.org/Team:BGIC-Union/Gold_Integrated">Integrated Human Practice</a></h4>
 
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<a href="https://2017.igem.org/Team:BGIC-Union/Model"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/48/Bgicmodel.png " width="400px" height="300px"></img></a>
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/2/21/Bgicpublicengagement.png " width="200px" height="150px"></img></a>
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<h4>Model</h4>
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<h4><a href="https://2017.igem.org/Team:BGIC-Union/Engagement">Education & Public Engagement</a></h4>
 
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<a href="https://2017.igem.org/Team:BGIC-Union/Demonstrate"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/f/f2/Bgicdemonstrate.png" width="400px" height="300px"></img></a>
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/7c/Entreprenuershipbgic.png" width="200px" height="150px"></img></a>
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<h4>Demonstrate</h4>
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<h4><a href="https://2017.igem.org/Team:BGIC-Union/Entreprenuership">Entreprenuership</a></h4>
 
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<a href="https://2017.igem.org/Team:BGIC-Union/Improve"><img class="img-responsive" src="https://static.igem.org/mediawiki/2017/2/21/Improvebgic.png" width="400px" height="300px"></img></a>
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/48/Bgicmodel.png " width="200px" height="150px"></img></a>
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<h4>Improve</h4>
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<h4><a href="https://2017.igem.org/Team:BGIC-Union/Model">Model</a></h4>
 
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                </section>
 
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                <!-- END OUR CLIENTS -->
 
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<div class="container">
 
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<<h2>ABSTRACT</h2>
 
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<h3>dCasentry: A guardian against Lung Cancer</h3>
 
−
Lung cancer is one of the deadliest malignant tumors in the world, with highest death rate in China. The most widely used lung cancer detection method nowadays remains biopsy, which can cause pain and other complication to patients. However, the technic of liquid biopsy obviates this problem by detecting various factors including circulating tumor DNA (ctDNA) in the blood. ctDNA is a short strand of DNA released from tumor cells that can be deemed as the sign of cancer. Although traditional liquid biopsy avoids pain, it requires laboratory apparatus such as PCR instrument during detection. Also, some other ctDNA detection systems fail to overcome the difficulty of the extremely low concentration of ctDNA in the blood. To solve those problems, we developed a new liquid biopsy method using dCas9 protein with a much simple procedure while increase the signal with various kinds of output. We hope to adopt free-dried paper chip as our vector of detecting system to simplify its operation as well as storage. Finally, the product will be presented as a kit that contains all the items you need for detection and can be easily used on field without any machines.
 
−
<h2>PROJECT</h2>
 
−
<div id= “description”>
 
−
<h3>Introduction</h3>
 
−
<Strong>Background</Strong>
 
−
Lung cancer is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. This growth can spread beyond the lung by the process of metastasis into nearby tissue or other parts of the body which can eventually cause death. The incidence rate of lung cancer is higher than any other common cancers in China. Among the ten most common cancers considered in the temporal trend analyses for men, lung cancer always has the highest incidence rates compares to others, with around 1 male per 2000 got lung cancer every year.
 
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<div class= “centered mainstyle” height=“600px”>
 
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<img src= “ https://static.igem.org/mediawiki/2017/0/0a/Cancerbgic.jpg” height= “400px” ></img>
 
−
<h6>Figure 1. Trends in Incidence Rates (Age-Standardized to the Segi Standard Population) for Selected Cancers for Males: China, 2000 to 2011.</h6>
 
−
</div>
 
−
<strong>ctDNA Detection</strong>
 
−
Nowadays, multiple lung cancer detection methods are developed. The most reliable and effective method is tissue biopsy. However, lung biopsy can cause pain and other complication such as Pneumothorax and bleeding. Therefore, people try to develop a new way for carrying out biopsy which minimized trauma and pain. Liquid biopsy, as the name implies, this process involving tumor markers detection in the blood from the patients to determine whether the patients have cancer. Unlike traditional biopsy, liquid biopsy does not require samples from patients' tumor, which exempted from the pain caused by biopsy in the traditional detection process. Nowadays, the common method to liquid biopsy has to detect circulating tumor DNA (ctDNA), circulating tumor cells (CTC) and secretion.
 
−
</br>
 
−
ctDNA, which stands for circulating tumor DNA, is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It is released by the dying cells during the biological process of apotheosis and necrosis, or active release from viable tumor cells.
 
−
 
−
<img src= “https://static.igem.org/mediawiki/2017/a/a8/Ctdnabgic.jpg” height= “400px” ></img>
 
−
<h6>Figure 2. Circulating tumor DNA (ctDNA) is released by tumor cells and enters the circulation system.</h6>
 
−
 
−
</br>
 
−
Although the liquid biopsy avoid complication, common liquid biopsy required laboratory apparatus such as PCR instrument during the detection of ctDNA. So we want to take this method further, to develop a new liquid biopsy method of detecting ctDNA with a much simple process.
 
−
</div>
 
−
<div id= “Design”>
 
−
<h3>Design</h3>
 
−
The first thing came into our mind is Cas9 system. But how can we apply Cas9 system into detecting ctDNA? In searching for solution, the project of Peking University IGEM in 2015 provided a lot enlightenments for our design.
 
−
<strong>Paired dCas9 design of Peking University and NUDT</strong>
 
−
Peking University IGEM team in 2015 dedicates to build a new reporter that is able to convert the sequence-specific information into easily readable signal including bioluminescence.[1] Compare to the previous nucleic acid detection methods such as q-PCR, probe method and direct sequencing, their method carries out a faster, less-risky and more specific detection.
 
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<div height= “700px”>
 
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<img src= “https://static.igem.org/mediawiki/2017/d/d6/Dcas9fkuc.jpg”></img>
 
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−
<h6>Figure 3. Schematic of the paired dCas9 (PC) Reporter system of Peking University.  A complex is formed by dCas9, split luciferase(Nluc or Cluc) and sgRNA. In the presence of target DNA, each of the complexes will bind to the specific sites indicated by sgRNA. When they approach to each other, the split luciferase will reassemble and generate bioluminescence signal.</h6></div>
 
−
</br>In order to increase the specificity and visualize the results, Peking University invents a programmable paired-dcas9 report system (PC report system) with luciferase. dCas9 is a variant of Cas9 protein that is catalytically dead. In other word, it can combine with sgRNA and lead itself to target site like Cas9 but it is unable to cleave DNA strand. The system designed by Peking is consisted of a pair of dcas9 which fused with split luciferase (Nluc and Cluc) respectively. Thus, two kinds of split luciferase-dCas9-sgRNA complexes can be formed. With the presence of target DNA, each of the complexes will bind with each complemented site. In this way, if the sgRNA is designed properly close, the two complexes will approach to each other so that two split luciferase attached to them will reassemble and generate bioluminescence signal.
 
−
</br>
 
−
Likewise, NUDT of IGEM 2016 gave similar design. [2] Although intended to detect microRNA, their split-HRP-dCas9 design can also be utilized to detect ctDNA. The reunion of split HRP will convert the presence of Target DNA into visual output signal by adding substrates such as 3,3',5,5'-Tetramethylbenzidine (TMB)
 
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<div class= “centered mainstyle” height=“600px”>
 
−
<img src= “https://static.igem.org/mediawiki/2017/a/aa/Mirna.jpg” ></img>
 
−
 
−
 
−
 
−
 
−
<h6>Figure 4. Schematic representation of split HRP PC report system of NUDT. Each fragments of split HRP(sHRPN and sHRPC) will bind to dCas9 protein, and they will together form a sHPR-dCas9-sgRNA complex. Like PC report system of Peking University, the split HRP will reunion in the presence of target gene and produce visual signal by adding substrates such as 3,3',5,5'-Tetramethylbenzidine (TMB).</h6></div>
 
−
</br>
 
−
However, there are still several possible improvements in those systems. First, due to its reliance on luciferase, it need other materials like fluorescein to function. Second, it is able to produce only one type of output -- bioluminescence. Third, which is the most important one, is that it cannot amplify signal. In order to deal with the extremely low concentration of ctDNA in blood, we invent our system similar to them using T7 polymerase.
 
−
 
−
<strong>Paired dCas9-T7 Report System</strong>
 
−
 
−
Paired dCas9-T7 report system is a more efficient and precise PC report system with flexible outputs and signal amplifying ability.
 
−
</br>
 
−
Like Peking University, we split the T7 polymerase and connect it with dCas9 protein via a linker. We call each complex NT7-dCas9 and CT7-dCas9.
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
<img src= “https://static.igem.org/mediawiki/2017/a/a7/T7re.png
 
−
” height= “400px” ></img>
 
−
 
−
<h6>Figure 5. The schematic illustration of paired dCas9 system(1). dCas9 is connected to one of the piece of split T7 RNA polymerase(NT7 and CT7). They will combine with sgRNA beforehand and constitute the split T7-dCas9-sgRNA complex.
 
−
</h6></div>
 
−
</br>
 
−
Each complex by itself is inactive, but when the two complex attached to particular sites we choose for identification of special sequences, they will reassemble to form a completed active T7 polymerase and start transcription in the presence of T7 promotor in cell free environment.
 
−
</br>
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
<img class="img-responsive"  src= “https://static.igem.org/mediawiki/2017/a/a5/Combine.png” height= “400px” ></img>
 
−
 
−
<h6>Figure 6. The schematic illustration of paired dCas9 system(2). With the presence of target DNA, each complex will bind with pre-designed sgRNA-binding site on the sequence. When the split T7 polymerase approach each other close enough, they will become active and start transcription by adding report gene with T7 promotor in cell free system. After transcription, the mRNA will be translated into report protein like GFP and RFP which generate signal output. </h6></div>
 
−
 
−
</br>
 
−
Our design has two major advantages over Peking University’s project. First, various types of output are possible. Because the reassembled protein is a RNA polymerase, it can convert ctDNA into different types of signals, including GFP, RFP, luciferase or LacZ with a T7 promotor and a following coding sequence. Besides, with the presence of Target sequence, reunion T7 polymerase can transcribe DNA into mRNA which will be further translated into protein consistently. Therefore, the signal can be magnified over time even if the initial ctDNA concentration is undesired.
 
−
 
−
 
−
<strong>Target sequence and sgRNA binding site</strong>.
 
−
After designing the T7 PC report system, we started to find a suitable gene locus. After a long time searching for gene locus, we finally chose the fused gene EML4-ALK.</br>
 
−
The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) is a fusion gene resulting from chromosome inversion and was identified in nonsmall cell lung cancer(NSCLC). (Wong DW) [3] The specific medicine, ALK inhibitor Crizotinib, has been reported to have curative effect on repression of the tumor and remission of relative symptom.[4]
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
 
−
 
 
−
<h6>Figure 7. Schematic representation of a fusion gene.</h6></div>
 
−
</br>
 
−
We decide to detect EML4-ALK because 1) it has effective specific drug. 2) Fusion gene can effectively avoid off-target effect of Cas9 system.
 
−
  </br>First, many types of ctDNA have already been detected but doctors only have drugs for few of them. Thus, detecting EML4-ALK whose specific drug, crizotinib, has been found, is more beneficial to patients than other ctDNA. Patients can have precise treatment as soon as possible when the detection result is positive.
 
−
  </br>Second, false-positive result can be evaded by detecting fusion gene. Sometimes dCas9 protein may recognize a very similar site as its target and it generates false positive result in this case. For example, the sgRNA may bind with complementary sequence with one or two incompatible base pair. However, the corresponding T7 polymerase will continue to reunion and give out positive output. In this way, our system will generate a lot false-positive result in detecting point mutation or SNP because it will confuse the normal sequence and cancer sequence with one or two mutations. However, fused gene can obviate this problem. Fused ctDNA composed of two fused normal DNA, which means that if we design our two sgRNA binding sites separately in two sequence or located in the part they connected to with each other, we can avoid off-target effect of the report system.
 
−
</br>
 
−
The EML4-ALK fused gene has two variants (A and B).
 
−
More details：
 
−
https://www.ncbi.nlm.nih.gov/nuccore/AB374361.1 EML4-ALK variant 3 a
 
−
https://www.ncbi.nlm.nih.gov/nuccore/AB374362.1 EML4-ALK variant 3 b
 
−
 
−
</br>We design our sgRNA binding site as the principles as mentioned – distributing two binding sites in each of two parts of the sequence separately. Also, there are other limits in selecting sgRNA binding site. First, it needs a PAM site next to it(NGG). Also, the distance between a pair of sites(SpaceDistance) should either too close or too distant(5-35 is desirable, as suggested by Peking University), so the choices of sites are further limited.
 
−
</br>First, we wrote a computer program to find all PAM site and find out of the combination with the distance between them.
 
−
</br>Computer stimulation for Variant A
 
−
<div height= “800px”>
 
−
 
−
 
−
<h6>Table 1. Computer stimulation result for possible sgRNA binding sites of EML4-ALK variant A and the distance between them.</h6>
 
−
</div>
 
−
 
−
</br>Computer stimulation for Variant B
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
 
−
 
−
<h6>Table 2. Computer stimulation result for possible sgRNA binding sites of EML4-ALK variant B and the distance between them.
 
−
</h6></div>
 
−
</br>Second, we choose four sgRNA binding sites in variant A and three sites in variant B. (23a 33a 83a 93a 70b 126b 147b) There are 7 combo in total, four in A and three in B.
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
 
−
 
−
<h6>Figure 8. The four sgRNA binding sites of fusion gene EML4-ALK Variant A. We designed four sgRNA binding sites in target gene – sgRNA23, sgRNA33 sgRNA83 and sgRNA93. The number of them indicates the location of their first base pair in target gene. The combination of sgRNA binding sites will be based on the rule that a pair of sgRNA should be distributed in EML4 or ALK area separately or one of them located on the connecting area of the EML4-ALK fusion gene.</h6>
 
−
 
−
 
−
<h6>Figure 9. The four sgRNA binding sites of fusion gene EML4-ALK Variant B. We designed three sgRNA binding sites in target gene – sgRNA70, sgRNA126 sgRNA147 and sgRNA93. The number of them indicates the location of their first base pair in target gene. The combination of sgRNA binding sites will be based on the rule that a pair of sgRNA should be distributed in EML4 or ALK area separately or one of them located on the connecting area of the EML4-ALK fusion gene.</h6>
 
−
</div>
 
−
 
−
 
−
</br>We intended to translate sgRNA in vitro. In order to achieve it, a cassette of T7-promotor-sgRNA-T7 terminator is indispensable and we planed to insert it into psb1c3 plasmid and submit them to IGEM registry as our new Biobricks.
 
−
<div class= “centered mainstyle” height=“600px”>
 
−
 
−
 
−
<h6> Figure 10. The plasmid of sgRNA generator. The sgRNA generator Biobrick is composed of a T7 promotor, coding sequence for sgRNA and a T7 terminator.
 
−
 
−
</br>We planned to use oligo DNA bridging to construct those sequences.
 
−
The construction scheme was acquired from DNAWorks, which is a website for “Automatic oligonucleotide design for PCR-based gene synthesis”. SOE-PCR
 
−
OligoDNA 的设计图
 
−
SOEPCR 原理图
 
−
 
−
  </br>Also since those sgRNA cassette are only different in 20bp of their target sequence, we guessed that we could simplify the experiments by find solutions given by DNAWorks by finding as many same oligo DNA in the solution of seven sgRNA as possible. So, we wrote a program to do it. Finally, we reduced the ordered oligo DNA from 42 to 24.
 
−
 
−
Shared oligo DNA 解释图
 
−
<
 
−
<h3>Experiment</he>
 
−
<strong>
 
−
1. Expression of PC report system</strong>
 
−
 
−
Construct expression system for T7 PC report system and Fluc PC report system.
 
−
</br>
 
−
T7 PC report system: </br>
 
−
 
−
<h6> Figure 1. Plasmid of NT7-dCas9 on pet28a backbone. Our expression plasmid on pet28a backbone of NT7-dCas9 includes a T7 promotor, a lac repressor, the NT7-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
 
−
 
−
<h6> Figure 2. Plasmid of CT7-dCas9 on pet28a backbone. Our expression plasmid of CT7-dCas9 on pet28a backbone includes a T7 promotor, a lac repressor, the CT7-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
 
−
 
−
Fluc PC report system:
 
−
 
−
<h6> Figure 3. Plasmid of Nluc-dCas9 on pet28a backbone. Our expression plasmid of Nluc-dCas9 on pet28a backbone includes a T7 promotor, a lac repressor, the Nfluc-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
 
−
 
−
 
−
<h6> Figure 4. Plasmid of Cfluc-dCas9 on pet28a backbone. Our expression plasmid of Cfluc-dCas9 on pet28a backbone includes a T7 promotor, a lac repressor, the Cfluc-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
 
−
 
−
 
−
酶切验证图
 
−
Method 给一个好的格式
 
−
解释为什么要做fluc
 
−
 
−
</br>We construct both two parts for T7 PC report system and Fluc PC report system. Fluc PC report system was created to testify its ability and compare its efficiency with ours.
 
−
</br>Each plasmid is constructed with a T7 promotor, a lac repressor, a RBS and the coding sequence followed by a 6 X His-Tag on PET28a backbone with kanamycin resistance gene. A T7 promotor and a T7 promotor, which can start and stop transcription in the presence with T7 RNA polymerase, are installed at start and end of the part respectively. Lac repressor is added for the induction of protein because it can silence the genes behind it unless IPTG is introduced. A 6 X polyhistidine–tag(His - Tag) is included after the coding sequence in order to purify the protein.
 
−
 
−
</br>Cloning:
 
−
</br>The coding sequence of T7 polymerase is acquired from the Eco.li BL21(DE3), whose genome is been genetically modified to include the coding sequence of T7 polymerase.
 
−
</br>The coding sequence of luciferase is acquired from the control DNA template, pBESTluc™ Vector containing the eukaryotic firefly luciferase gene in E. coli S30 Extract System for Circular DNA, Promega.
 
−
</br>The coding sequence of dCas9 is acquired from the plasmid pX335-dCas9 plasmid ordered from Addgene.
 
−
</br>Half of the linker is adhered to the end of NT7,CT7,Nfluc, Cfluc, and half of linker is adhered to the start of dCas9 by modification of 5’ end in primers of PCR. In order to connect the sequence more easily, we rewrite the coding sequence of Linker from GGGGSGGGGS into GGGGGSGGGS in order to create a BamHI restrictive endonuclease site.
 
−
</br>Likewise, the His-Tag sequence is also adhered to end of dCas9 by modification of 5’ end in primers of PCR.
 
−
</br>Then the NT7-dCas9-his, CT7-dCas9-his, Nfluc-dCas9-his and Cfluc-dCas9-his coding sequences are inserted into a PET28a plasmid with T7 promotor, lac repressor, RBS and T7 terminator by Golden Gate Assembly.
 
−
 
−
 
−
 
−
</br>Protein induction and purification
 
−
</br>To analyze the function the PC report system, the target protein should be induced and purified from bacteria
 
−
.
 
−
</br>For comparison, we ask plasmids of HRP PC report system from NUDT.
 
−
 
−
</br>In order to induce the protein, we first transform all the pET28a plasmids into Eco.li strain BL21(DE3), whose genome is genetically modified to contain the coding sequence for T7 polymerase. After preserving the bacteria, we underwent the following procedures.
 
−
 
−
</br>Pre-Culture
 
−
</br>Inoculate all the preserved bacteria into 5ml of LB broth.
 
−
</br>Incubate the samples in the shaker for one night.
 
−
 
−
</br>Seed-Culture
 
−
</br>Inoculate 20ul previous bacteria liquid into new tubes with 5ml of LB broth. Measure and record the OD600 value of each sample in the Nanodrop. In order to find out the best IPTG concentration for induction, we tested a gradient of IPTG concentration. When the OD600 value reaches 0.4 – 0.6, add appropriate amount of IPTG so that the final concentration is in the gradient of 100uM, 200uM, 300uM, 400uM.
 
−
</br>Incubate the samples in the shaker in 24 degrees centigrade for 19 hours.
 
−
 
−
</br>We then conducted the extraction of protein using a commercial Kit.( xTractor Buffer Kit, Clontech). We centrifuged samples to collect the bacteria pellet and resuspend them in buffer provided by the kit together with lysozyme and DNase I. The lysate with target protein was collected from the centrifuged cell debris. Then we resuspend the cell debris in the same buffer and same volume with clear lysate. Then a series of polyacrylamide gel electrophoresis(PAGE) were conducted to find out the best induction concentration. Sometime, when the concentration of IPTG is too high, excessive amount of target protein will force bacteria to transform them into inclusion body protein which can not be purified. Thus the rule of thumb is to find out the concentration with least target protein in cell debris solution and with highest target protein in supernate. All of the four protein, their molecular weight is around 200KD. With all the four concentration(100uM, 200uM, 300uM, 400uM), we concluded that 200uM is the best induction concentration for four of them.
 
−
 
−
 
−
<h6> Figure 5. PAGE of Nfluc-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
 
−
 
−
 
−
 
−
 
−
 
−
<h6> Figure 6. PAGE of Cfluc-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
 
−
 
−
 
−
<h6> Figure 7. PAGE of NT7-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
 
−
 
−
 
−
<h6> Figure 8. PAGE of CT7-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
 
−
 
−
</br>After determining that we have successfully induced all of the four proteins, we adopted a commercial kit to purify the samples. (Capturem™ His-Tagged Purification Miniprep Kit, Clontech)
 
−
 
−
</br>His-Tag is a series of histidine amino acids that can combine itself with the nickel column of tube provided by the kit. By this means, the target protein with his-tag can be separated from Flowthrough. Then we used elution buffer containing imidazole to wash the nickel column to collect target protein.
 
−
A PAGE was conducted to confirm the efficiency of purification.
 
−
 
−
<h6> Figure 9. The PAGE of NT7-dCas9, CT7-dCas9, Nfluc-dCas9,Cfluc-dCas9 purified solution and Flowthrough. The red circle indicated the expected protein.
 
−
 
−
</br>As the <h6> Figure shows, we have successfully purified the protein.
 
−
 
−
</br>However, proteins cannot work with the presence of imidazole. We therefore use a commercial kit to conduct a buffer exchange(Pierce™ Protein Concentrators PES, 30K MWCO, Thermo Fisher). After turns of wash, the protein were transferred from imidazole solution to working solution (20 mM HEPES, 150 mM KCl, pH 7.5).
 
−
 
−
<strong>
 
−
2. Production and purification of RNA</strong>
 
−
sgRNA guides the dCas9 protein to its destination. We intended to translate sgRNA in vitro. In order to achieve it, a cassette of T7-promotor-sgRNA-T7 terminator is indispensable and we planed to insert it into psb1c3 plasmid and submit them to IGEM registry as our new Biobricks.
 
−
</br>
 
−
Constructing expression system for T7 promotor – sgRNA – T7 terminator cassette </br>
 
−
A sgRNA comprises of a 20bp guiding sequence and a 77bp scaffold sequence. Together with a T7 promotor and T7 terminator and restrictive enzyme site prepared for insertion, the length for each sgRNA is 213bp.
 
−
 
−
<h6> Figure 10. The plasmid of sgRNA generator. The sgRNA generator Biobrick is composed of a T7 promotor, coding sequence for sgRNA and a T7 terminator.</h6>
 
−
</br>
 
−
First, we mixed all the six oligo DNAs with PCR mix. A standard PCR was conducted with the annealing temperature suggested by DNAWorks.
 
−
Second, the outer primers was added to the purified product of the first step.
 
−
Finally, run agarose gel analysis and collect the product.
 
−
</br>
 
−
After restrictive enzyme digestion, all of the seven sgRNA were inserted into psb1c3 plasmid. All of the plasmids were then transformed in to Eco.li strain DH5a and amplified. Sequencing of the samples was done by BGI and most of them were successful.
 
−
</br>
 
−
We also conducted restrictive enzyme check and PCR check to determine its accuracy.
 
−
<div class= “centered mainstyle” height=“600px”>
 
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<h6> Figure 11. Restrictive enzyme check.(XbaI and Spel) Restrictive enzyme pair XbaI and Spel were used to conduct the enzyme check. The Expected length is around 200bp and all the length should be the same.</h6>
 
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差一张PCR鉴定的图
 
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The in vitro transcription and purification of sgRNA.
 
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We used MEGAscript™ T7 Transcription Kit to yield sgRNA in vitro.
 
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A PCR was conducted to obtain the linear T7 promotor – sgRNA – T7 terminator cassette. The PCR product was then mixed with T7 RNA polymerase, ribonucleotides and reaction buffer.
 
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After six hours of incubation at 37 degrees centigrade, DNase was introduced to remove the template DNA.
 
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</br>
 
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We used a commercial miRNA purification kit to separate the transcribed sgRNA.(Ambion® mirVana™ miRNA Isolation Kit)
 
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After turns of wash and purification, the final RNA product diluted in nuclease-free water can reach the concentration of 1500 ng/ul or higher.
 
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</br>
 
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Also, a RNA electrophoresis was conducted to examine the existence of sgRNA.
 
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RNA的胶图
 
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The sgRNA samples were then stored in -20 degrees centigrade.
 
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3. NASBA
 
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Part:
 
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Biobricks:
 
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We create four Basic Biobricks regarding to T7-dCas9.
 
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NT7-dCas9 BBa_K2371000
 
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CT7-dCas9 BBa_K2371001
 
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NT7      BBa_K2371002
 
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CT7      BBa_K2371003
 
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Part的部分能否做成可折叠的
 
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Description:
 
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The first two Biobricks are the two split T7 polymerase sequence we adopted. We provide them for other team to leverage them through other methods. We choose the split site suggested by the team of Tiyun Han, Quan Chen and Haiyan Liu. [5]
 
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The following two Biobricks are the sequence of T7 polymerase connected to dCas9 protein. The linker between split T7 polymerase and dCas9 is GGGGSGGGGS adopted by Peking University. A 6 X His-tag is added on C terminus of dCas9 in order to purify it.
 
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We create seven composite Biobricks regarding to sgRNA.
 
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We created a Biobrick for each of sgRNA and insert T7 promotor and T7 terminator before and after the sequence respectively in order to utilize them to transcribe sgRNA in vitro. The sequences are synthesize through Oligo DNA bridging designed by DNAworks. The detail of synthesis method is in the
 
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experiment.
 
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<h6> Figure 10. The plasmid of sgRNA generator. The sgRNA generator Biobrick is composed of a T7 promotor, coding sequence for sgRNA and a T7 terminator.
 
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sgRNA generator for EML4-ALK Variant A 23  BBa_K2371004
 
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sgRNA generator for EML4-ALK Variant A 33  BBa_K2371005
 
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sgRNA generator for EML4-ALK Variant A 83  BBa_K2371006
 
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sgRNA generator for EML4-ALK Variant A 93  BBa_K2371007
 
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sgRNA generator for EML4-ALK Variant B 70  BBa_K2371008
 
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sgRNA generator for EML4-ALK Variant B 126 BBa_K2371009
 
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sgRNA generator for EML4-ALK Variant B 147 BBa_K2371010
 
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We create two supplemental composite biobricks for expression of GFP and RFP in cell free system.
 
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pT7-eforRed BBa_K2371011
 
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pT7-amilGFP BBa_K2371012
 
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These two biobricks are composed of a T7 promotor with a coding sequence of GFP or RFP.
 
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These two biobricks are supposed to cooperate with T7 PC report system in cell free system. When the split T7 polymerase reassemble, it will start transcription of GFP and RFP from these two plasmids.
 
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GFP 和 RFP 的biobrick质粒图
 
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Reference:
 
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1. Yihao,Z.et al.,2017.Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains. ACS Synth. Biol., 2017, 6 (2), pp 211–216.
 
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2. Development of A NovelBlood-MicroRNA Handy Detection System with CRISPR. https://2016.igem.org/Team:NUDT_CHINA
 
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3. Wong DW, et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer. 2009 Apr 15;115(8):1723-33.
 
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4. Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014; 371 :2167-77
 
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5. Tiyun,H.et al.,2016.Engineered photoactivatable genetic switches based on the bacterium phage T7 RNA polymerase.ACS Synthetic Biology,http://pubs.acs.org on October 31, 2016.
 
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Latest revision as of 13:11, 2 December 2017

Team：BGIC-Union

Hi, I am dCasentry,

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