Difference between revisions of "Team:FAFU-CHINA"

 
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<meta charset="utf-8">
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<meta name="viewport" content="width=device-width, maximum-scale=1">
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<head>
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<title>Homepage</title>
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<link rel="icon" href="https://static.igem.org/mediawiki/2015/1/19/Fafu_favicon.png" type="image/png">
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<link rel="stylesheet" type="text/css" href="https://static.igem.org/mediawiki/2014/e/ea/Rutgers_normalize.txt" />
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<link rel="shortcut icon" href="favicon.ico" type="img/x-icon">
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  <script src="https://static.igem.org/mediawiki/2014/5/56/Rutgers_modernizr.txt"></script>
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/wailian/css/Montserrat?action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/wailian/css/Open-Sans?action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" href="https://2015.igem.org/Template:FAFU_CHINA/css/homecss?action=raw&amp;ctype=text/css" type="text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/bootstrap?
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action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/style?
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action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/stylesheet?
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action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/font-awesome?
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action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/responsive?
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action=raw&amp;ctype=text/css" />
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<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/index2/css/animate?
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action=raw&amp;ctype=text/css" />
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<script type="text/javascript"  
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src="http://https://2015.igem.org/Template:FAFU_CHINA/index2/js/jquery-1-8-3-min?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript" src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/bootstrap?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript" src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/jquery-scrolltofixed?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript"
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src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/jquery-easing-1-3?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript"
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src="http://https://2015.igem.org/Template:FAFU_CHINA/index2/js/jquery-isotope?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript" src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/wow?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript" src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/classie?action=raw&amp;ctype=text/javascript"></script>
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    <script src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/respond-1-1-0-min?action=raw&amp;ctype=text/javascript"></script>
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    <script src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/html5shiv?action=raw&amp;ctype=text/javascript"></script>
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    <script src="https://2015.igem.org/Template:FAFU_CHINA/index2/js/html5element?action=raw&amp;ctype=text/javascript"></script>
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<![endif]-->
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</head>
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<body>
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<div style="overflow:hidden;">
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<header class="header" id="header"><!--header-start-->
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<div class="container">
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    <figure class="logo animated fadeInDown delay-07s">
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        <a href="#"><img src="https://static.igem.org/mediawiki/2015/b/b7/FAFU-CHINA_Silencer_new.png" alt=""></a>
 +
        </figure>
 +
        <h1 class="animated fadeInDown delay-07s">Welcome to FAFU-CHINA iGEM</h1>
 +
        <ul class="we-create animated fadeInUp delay-1s">
 +
        <li style=“color:#ffffff;font-size:36px;”>CSBV.Silencer</li>
 +
        </ul>
 +
        <br>
 +
            <a class="link animated fadeInUp delay-1s" href="#">Get Started</a>
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    </div>
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</div>
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</header><!--header-end-->
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 +
 
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<nav class="main-nav-outer" id="test"><!--main-nav-start-->
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<div class="container">
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        <ul class="main-nav">
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        <li><a href="#header">Home</a></li>
 +
                <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Attributions">Attributions</a></li>
 +
                <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Team">Team</a></li>
 +
                <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Practices ">Human practice</a></li>
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                <li class="small-logo"><a href="https://2015.igem.org/Team:FAFU-CHINA"><img src="https://static.igem.org/mediawiki/2015/1/13/Fafu_small_logo.png" alt=""></a></li>
 +
            <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Project">Project</a></li>
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            <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Parts">Parts</a></li>
 +
            <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Safety">Safety</a></li>
 +
         
 +
         
 +
            <li><a href="https://2015.igem.org/Team:FAFU-CHINA/Collaborations">Collaborations</a></li>
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        </ul>
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<a class="res-nav_click" href="#"><i class="fa-bars">BACK TO HOME</i></a>
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    </div>
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</nav><!--main-nav-end-->
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<section class="main-section paddind" id="Portfolio"><!--main-section-start-->
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<div class="container">
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    <h2>&nbsp; &nbsp;&nbsp;Project</h2>
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      <div class="portfolioFilter"> 
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        <ul class="Portfolio-nav wow fadeIn delay-02s">
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        <li><a href="#" data-filter="*" class="current" >Back To Header</a></li>
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        </ul>
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      </div>
 +
       
 +
</div>
 +
    <div class="portfolioContainer wow fadeInUp delay-04s">
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            <div class=" Portfolio-box printdesign">
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                <a href="https://2015.igem.org/Team:FAFU-CHINA/Design"><img src="https://static.igem.org/mediawiki/2015/9/9d/FAFU-CHINA_Small_logo_%283%29.jpg"
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 +
alt=""></a>
 +
               
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                    <p>Design</p>
 +
                </div>
 +
                <div class="Portfolio-box webdesign">
 +
                <a href="https://2015.igem.org/Team:FAFU-CHINA/Meeting"><img src="https://static.igem.org/mediawiki/2015/a/ab/FAFU-CHINA_Small_logo_1_%284%29.jpg"
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alt=""></a>
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                    <p>Meeting</p>
 +
                </div>
 +
                <div class=" Portfolio-box branding">
 +
                <a href="https://2015.igem.org/Team:FAFU-CHINA/Notebook"><img src="https://static.igem.org/mediawiki/2015/e/e5/FAFU-CHINA_Small_logo_1_%285%29.jpg"  
 +
 
 +
alt=""></a>
 +
               
 +
                    <p>Notebook</p>
 +
                </div>
 +
                <div class=" Portfolio-box photography" >
 +
                <a href="https://2015.igem.org/Team:FAFU-CHINA/Protocol"><img src="https://static.igem.org/mediawiki/2015/f/f4/FAFU-CHINA_Small_logo_1_%287%29.jpg"
 +
 
 +
alt=""></a>
 +
               
 +
                    <p>Protocol</p>
 +
                </div>
 +
                <div class=" Portfolio-box branding">
 +
                <a href="https://2015.igem.org/Team:FAFU-CHINA/Results"><img src="https://static.igem.org/mediawiki/2015/3/3e/FAFU-CHINA_Small_logo_1_%288%29.jpg"
 +
 
 +
alt=""></a>
 +
               
 +
                    <p>Result</p>
 +
                </div>
 +
                <div class=" Portfolio-box photography">
 +
                <a href="https://2015.igem.org/Team:FAFU-CHINA/Project#Theory"><img src="https://static.igem.org/mediawiki/2015/4/42/FAFU-CHINA_Small_logo_1_%2811%29.jpg"
 +
 
 +
alt=""></a>
 +
               
 +
                    <p>Theory</p>
 +
                </div>
 +
    </div>
 +
</section><!--main-section-end-->
 +
 
 +
<section class="main-section" id="service"><!--main-section-start-->
 +
<div class="container">
 +
            <div class="row">
 +
              <div class="col-lg-12 col-sm-12 wow fadeInLeft delay-05s">
 +
                          <h2>Safety & Part</h2>
 +
                </div>
 +
              </div>
 +
        <div class="col-lg-4 col-sm-6 wow fadeInLeft delay-05s">
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            <div class="service-list">
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                <div class="service-list-col1">
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                    <i class="fa-paw"></i>
 +
                    </div>
 +
                <div class="service-list-col2">
 +
                        <h2>safety</h2>
 +
                        <p>Bio-safety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health. These prevention mechanisms include conduction of regular reviews of the bio-safety in laboratory settings, as well as strict guidelines to follow. </p>
 +
                        <br>
 +
                        <a href="https://2015.igem.org/Team:FAFU-CHINA/Safety">MORE</a>
 +
                        <br>
 +
                        <h1> Organisms we used :</h1>
 +
                    </div>
 +
                </div>
 +
                            </div>
 +
            <figure class="col-lg-8 col-sm-6  text-right wow fadeInUp delay-02s">
 +
            <img src="https://static.igem.org/mediawiki/2015/4/45/Fafu_safety.png" alt="">
 +
            </figure>
 +
       
 +
        </div>
 +
</div>
 +
</section><!--main-section-end-->
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<section class="main-section alabaster"><!--main-section alabaster-start-->
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<div class="container">
 +
    <div class="row">
 +
 
 +
<figure class="col-lg-5 col-sm-4 wow fadeInLeft">
 +
            <img  src="https://static.igem.org/mediawiki/2015/b/b8/FAFU-CHINA_improvement_part.png"
 +
 
 +
alt="">
 +
            </figure>
 +
<figure class="col-lg-5 col-sm-4 wow fadeInLeft">
 +
            <img  src="https://static.igem.org/mediawiki/2015/3/34/FAFU-CHINA_NEW_PART.png" alt="">
 +
            </figure></p>
 +
 
 +
        </div>
 +
</div>
 +
</section><!--main-section alabaster-end-->
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<!------------------此处移除1-------------->
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<section class="main-section client-part" id="client"><!--main-section client-part-start-->
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<div class="container">
 +
 
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    <div class="row">
 +
        <div class="col-lg-12">
 +
            <a href=" https://2015.igem.org/Team:FAFU-CHINA/Attributions"><p class="client-part-haead wow fadeInDown delay-05">Without everyone's participation,our igem team would still be a plan....</p></a>
 +
          </div>
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          </div>
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<style>
 +
p1{color:white;}
 +
</style>
 +
    <ul class="client wow fadeIn delay-05s">
 +
        <li><a href="#">
 +
            <img src="https://static.igem.org/mediawiki/2015/a/a4/Fafu_aaa.jpg" alt="">
 +
                <h1><p1>ATTRIBUTIONS</p1></h1>
 +
            </a></li>
 +
        </ul>
 +
    </div>
 +
</section><!--main-section client-part-end-->
 +
<div class="c-logo-part"><!--c-logo-part-start-->
 +
<div class="container">
 +
    <ul>
 +
 
 +
    </ul>
 +
</div>
 +
</div><!--c-logo-part-end-->
 +
<section class="main-section team" id="team"><!--main-section team-start-->
 +
<div class="container">
 +
        <div class="rightbxti"><a href="https://2015.igem.org/Team:FAFU-CHINA/Team"><h2>Team</h2></a></div>
 +
 
 +
 
 +
        <div class="team-leader-block clearfix">
 +
          <div style="border-left:400px;" class="team-leader-box">
 +
                <div style="border-left:400px;" class="team-leader wow fadeInDown delay-09s">
 +
                    <div style="border-left:400px;" class="team-leader-shadow"><a href="#"></a></div>
 +
                    <img src="https://static.igem.org/mediawiki/2015/7/7d/FAFU-CHINA_Team_members_%2811%29.png" alt="">
 +
                    <ul>
 +
                        <li><a href="#" class="fa-twitter"></a></li>
 +
                        <li><a href="#" class="fa-facebook"></a></li>
 +
                        <li><a href="#" class="fa-pinterest"></a></li>
 +
                        <li><a href="#" class="fa-google-plus"></a></li>
 +
                    </ul>
 +
                </div>
 +
                <h3 class="wow fadeInDown delay-09s">Xiaolei Huang</h3>
 +
                <span class="wow fadeInDown delay-09s">Instructor</span>
 +
          </div>
 +
 
 +
<!-->
 +
        </div>
 +
        <div class="team-leader-block clearfix">
 +
            <div class="team-leader-box">
 +
                <div class="team-leader wow fadeInDown delay-03s">
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                    <div class="team-leader-shadow"><a href="#"></a></div>
 +
                    <img src="https://static.igem.org/mediawiki/2015/9/93/FAFU-CHINA_Team_members_%2815%29.png " alt="">
 +
                    <ul>
 +
                        <li><a href="#" class="fa-twitter"></a></li>
 +
                        <li><a href="#" class="fa-facebook"></a></li>
 +
                        <li><a href="#" class="fa-pinterest"></a></li>
 +
                        <li><a href="#" class="fa-google-plus"></a></li>
 +
                    </ul>
 +
                </div>
 +
                <h3 class="wow fadeInDown delay-03s">Zujian Wu</h3>
 +
                <span class="wow fadeInDown delay-03s">Advisor</span>
 +
 
 +
            </div>
 +
         
 +
            <div class="team-leader-box">
 +
                <div class="team-leader wow fadeInDown delay-03s">
 +
                    <div class="team-leader-shadow"><a href="#"></a></div>
 +
                    <img src="https://static.igem.org/mediawiki/2015/a/ab/FAFU-CHINA_Team_members_%2814%29.png " alt="">
 +
                    <ul>
 +
                        <li><a href="#" class="fa-twitter"></a></li>
 +
                        <li><a href="#" class="fa-facebook"></a></li>
 +
                        <li><a href="#" class="fa-pinterest"></a></li>
 +
                        <li><a href="#" class="fa-google-plus"></a></li>
 +
                    </ul>
 +
                </div>
 +
                <h3 class="wow fadeInDown delay-03s">Taiyun Wei</h3>
 +
                <span class="wow fadeInDown delay-03s">Advisor</span>
 +
 
 +
            </div>
 +
            <div class="team-leader-box">
 +
                <div class="team-leader  wow fadeInDown delay-06s">
 +
                    <div class="team-leader-shadow"><a href="#"></a></div>
 +
                    <img src="https://static.igem.org/mediawiki/2015/2/21/FAFU-CHINA_Team_members_%2813%29.png" alt="">
 +
                    <ul>
 +
                        <li><a href="#" class="fa-twitter"></a></li>
 +
                        <li><a href="#" class="fa-facebook"></a></li>
 +
                        <li><a href="#" class="fa-pinterest"></a></li>
 +
                        <li><a href="#" class="fa-google-plus"></a></li>
 +
                    </ul>
 +
                </div>
 +
                <h3 class="wow fadeInDown delay-06s">Bingfeng Zhou</h3>
 +
                <span class="wow fadeInDown delay-06s">Advisor</span>
  
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</head>
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            </div>
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<body>
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−
<div class="container">
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<img class="RU_logo" src="https://static.igem.org/mediawiki/2014/a/ac/Rutgers_top_logo.png" />
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<section>
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<div class="tabs tabs-style-circlefill">
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<nav>
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<ul>
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<li><a href="#project-section"><span>Project</span></a></li>
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<li><a href="#team-section"><span>Team</span></a></li>
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<li><a href="#notebook-section"><span>Notebook</span></a></li>
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<li><a href="#safety-section"><span>Safety</span></a></li>
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<a href="https://igem.org"><img src="https://static.igem.org/mediawiki/2014/1/1a/Rutgers_igem_logo.png" width="50" class="iGEM_logo"/></a>
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</ul>
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</nav>
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<div class="content-wrap">
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            <div class="team-leader-box">
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<section id="project-section">
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                <div class="team-leader wow fadeInDown delay-03s">  
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<h2>The Next Step in DNA Synthesis</h2>
+
                    <div class="team-leader-shadow"><a href="#"></a></div>
 +
                    <img src="https://static.igem.org/mediawiki/2015/0/05/FAFU-CHINA_Team_members_%285%29.png "">
 +
                    <ul>
 +
                        <li><a href="#" class="fa-twitter"></a></li>
 +
                        <li><a href="#" class="fa-facebook"></a></li>
 +
                        <li><a href="#" class="fa-pinterest"></a></li>
 +
                        <li><a href="#" class="fa-google-plus"></a></li>
 +
                    </ul>
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                </div>
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                <h3 class="wow fadeInDown delay-03s">Penny</h3>
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                <span class="wow fadeInDown delay-03s">Advisor</span>
  
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<div class="fp"><p><i>De novo</i> DNA Synthesis is extremely important to the world of Synthetic Biology, but the low efficiency of today's DNA Synthesis technology limits us.</p></div>
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                <h3 class="wow fadeInDown delay-06s">Wenxu Wu</h3>
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                <span class="wow fadeInDown delay-06s">Web Advisor</span>
  
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<div class="fp"><p>We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the <a href="http://blog.ginkgobioworks.com/2012/01/14/commercial-gene-synthesis/">time</a> and <a href="http://synbiobeta.com/time-new-dna-synthesis-sequencing-cost-curves-rob-carlson/">cost</a> required. This needs to change.</p></div>
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                <h3 class="wow fadeInDown delay-09s">Mucheng Cai</h3>
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                <span class="wow fadeInDown delay-09s">Dry Lab</span>
  
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                <h3 class="wow fadeInDown delay-03s">Ruicheng Dai</h3>
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                <span class="wow fadeInDown delay-03s">Captain </span>
  
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<h2>A Primer on DNA Synthesis</h2>
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                <h3 class="wow fadeInDown delay-06s">Changlong Lu</h3>
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                <span class="wow fadeInDown delay-06s">Experiment Designer</span>
  
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<div class="fp"><p>Currently we use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even <a href="http://syntheticyeast.org/sc2-0/introduction/">genome construction</a>. The process looks something like this:</p></div>
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                <h3 class="wow fadeInDown delay-09s">Junhao Lu</h3>
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                <span class="wow fadeInDown delay-09s">Vice-Captain</span>
  
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<div class="fp"><p>This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that <a href="http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Synthetic_cycle">each of the chemicals depicted</a> <small>(trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile)</small> causes some <a href="http://www.glenresearch.com/GlenReports/GR21-211.html">harm</a> to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.</p></div>
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                <h3 class="wow fadeInDown delay-03s">Zhili Lai</h3>
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                <span class="wow fadeInDown delay-03s">Lecturer</span>
  
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<div class="fp"><p>The process pictured above typically causes enough side-reactions to chemically ruin 1.5% of the existing DNA strands in each and every cycle (leaving 98.5% of them intact and with the correct sequence). This concept is called <a href="http://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html#7">coupling efficiency</a>, and it's the reason we can only synthesize up to <a href="http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/faqs.html#11">130</a>-150 bases at a time (or even <a href="http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos">200</a> bases if you're willing to pay a whole lot extra).</p></div>
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                <h3 class="wow fadeInDown delay-06s">LIANGDI CHEN</h3>
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                <span class="wow fadeInDown delay-06s">Web Designer</span>
  
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                <h3 class="wow fadeInDown delay-09s">Tengzhou Huang</h3>
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                <span class="wow fadeInDown delay-09s">Dry Lab</span>
  
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<h2>Our Idea: Use Enzymes</h2>
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                <h3 class="wow fadeInDown delay-03s">Zirui Cheng</h3>
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                <span class="wow fadeInDown delay-03s">Dry Lab</span>
  
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<div class="fp"><p>We believe that both cost and efficiency could be vastly improved by using enzymes for <i>de novo</i> DNA Synthesis. Polymerases have evolved alongside nucleic acids for billions of years and developed beautiful two-metal-ion machinery that makes <a href="http://www.ncbi.nlm.nih.gov/pubmed/2434996">phosphate diester formation</a> a piece of cake. In theory, this machinery could be applied to <i>de novo</i> DNA Synthesis to eliminate side-reactions and significantly simplify the process overall. A more natural, aqueous synthesis environment would lend itself to higher efficiency, too.</p></div>
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                <h3 class="wow fadeInDown delay-06s">Yifan Tang</h3>
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                <span class="wow fadeInDown delay-06s">Dry Lab</span>
  
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<div class="fp"><p>This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs.</p></div>
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                <h3 class="wow fadeInDown delay-09s">Tong Tong</h3>
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                <span class="wow fadeInDown delay-09s">Data Analyst</span>
  
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<div class="fp"><p>This is the enzymatic process that our team has envisioned. The enzyme is a template-independent polymerase called <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846215/">Terminal deoxynucleotidyl Transferase</a> (TdT). It favors single-stranded 3' ends when it adds whatever dNTP's are available. Using the same principles as today's synthesis strategies, it could serve to catalyze the coupling step in each cycle. Upon inspection of this polymerase's binding pocket, it is apparent that even large blocking groups could fit snuggly without hindering the catalytic machinery, but we decided to test a small, simple blocking group for this project to begin with: the acetyl group.</p></div>
 
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<div class="fp"><p>We found that the acetyl group undergoes a bit of background hydrolysis, especially at high pH, so it wouldn't be the ideal blocking group. It does, however, perform well enough to make benchmarking possible so that we can establish a proof of principle for this strategy. We ran many assays, outlined below.</p></div>
 
  
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<h2>Accomplishments</h2>
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<div class="fp"><p>These assays serve to elucidate the functionality of TdT in the conditions of our envisioned DNA Synthesis strategy. We used short, single-stranded primers (each composed of 15 thymidine residues) with 5' fluorescent tags (5'-FAM), and visualized the results on 22% polyacrylamide nondenaturing gels. The images are oriented with the loading wells above, so that the shortest oligos (which travel the fastest) end up furthest down in each lane. Each assay has a Control lane containing only the 15-length primer for reference. All lanes are labeled with neon-green text superimposed over the strange gray blotches of loading dye.</p></div>
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<li><strong>Tested the extension of single-stranded primers with TdT</strong></li>
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<li>The lanes are labeled with how many minutes the primers were incubated with TdT and dTTP.</li>
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<li>This assay established that TdT will add ordinary (not blocked) thymidine triphosphate to the ends of the single-stranded primers. The 30-minute result has the longest oligos (having traveled the least distance), but it appears more condensed because it had less distance to effectively resolve the differing oligo lengths. In later assays we let the gels run as far as possible to avoid the bunching-up of long oligos.</li>
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<li>Based on data in the literature, we estimate that a few hundred bases were added to each oligo (on average) under these reaction conditions (detailed in the Notebook->Protocols section). A DNA ladder would unfortunately be pretty expensive to create for these short, single-stranded, fluorescent primers, so we only interpret the relative activity of the enzyme between different lanes. This assay method doesn't allow us to quantify activity.</li>
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                <h2><i class=" icon-map-marker"></i>Description:</h2>
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                <a href="https://2015.igem.org/Team:FAFU-CHINA/Practices"><span>To remove the barrier between science and normal people’s life, It is our responsibility to lead people into the wonderland of science. This is a lofty ideal, and thousands of people in many fields have already been working for it for years. But this time, we need some innovations, perhaps some funs too.<br>Play is not only a simple process. We believe that play is actually a way of being, a way of learning, a way of doing science. So now If u ever watched the TED of Beau Lotto . You’ll know that play provides everything you need in oder to be a scientist, and that is what motivates us. If we add rules to play, we have a game which is actually what project is. And who can be better than children to play? Then we tried to spread the idea that everyone has the potential to be scientist as long as being offered an opportunity. Instead of giving a dull lesson about biology, this year FAFU-China decided to teach children how to play correctly, how to play with principles from science, and finally they will find their own way to science.... </span></a><br>Contact us:<br>Email:tripletau@126.com<br>Zipcode:350000
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<li><strong>Tested the effect of pH on acetylated thymidine incorporation</strong></li>
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<li>The lanes are labeled with a number representing the pH that the reaction was run in. Different MOPS buffers were used for the different pH's, except the last lane, which used NEB's Tris buffer (pH 7.9). Each reaction was carried out for 10 minutes with primers, TdT, and acetylated thymidine monomers (specific conditions can be found in the Notebook->Protocols section). The last lane used unmodified thymidine triphosphate, as a reference. </li>
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<li>This assay demonstrates that TdT adds less 3'-acetylated ("blocked") nucleotides at a lower pH. Aside from lane "7.0" (the anomaly), there is a clean step-ladder effect where longer oligos are produced at higher pH. There is still a significant spread of differing oligo lengths in the pH 6.5 lane, which indicates that enough hydrolysis occurs (of the acetyl blocking group) to allow for multiple additions. This conflicts with the findings of the later assays (below) that show no such spread of oligo lengths after reaction at pH 6.5.</li>
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<li>In other (unlisted) assays, we established that TdT gets progressively less active at pH's lower than 7.9. The "dTTP+NEB Buffer" lane shows the activity of TdT with unmodified nucleotides at pH 7.9. Comparing the activity in this lane with the others shows that acetylated nucleotides cause some truncation throughout the distribution of oligo lengths. Another (unlisted) assay shows that the difference in TdT's activity between NEB's Tris buffer and a homemade MOPS buffer (both at pH 7.9) was indiscernible.</li>
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<li><strong>Tested the extent of hydrolysis of incorporated acetylated thymidines</strong></li>
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<li>The lanes are labeled according to their respective pH of reaction. Labels ending in "a" were carried out with acetylated thymidine for 10 minutes, before splitting the reaction volume in half and halting the reaction in the first half. The second half continues to react, with more unmodified thymidine added to the mixture. The two lanes labeled "ccc" were similar reactions, except that they had unmodified thymidine in the first reaction ("a"). These "ccc" reactions were carried out at a pH of 6.5 to show how active the enzyme is at that pH (with unmodified nucleotides).</li>
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<li>This assay attempted to show that 3'-blocked nucleotides can effectively halt multiple additions, because of the terminal acetyl group. There is clearly a large amount of blocking-group-hydrolysis in the pH 7.9 reactions, leading to multiple additions when the extra unmodified nucleotide was added. The hydrolysis is limited enough at a pH of 6.5 that no significant amount of multiple additions occurs, even after adding extra unmodified nucleotide. These results are promising.</li>
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<li>The second "7.9a" lane in the bottom image is mislabeled, it should read "7.9b".</li>
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      <a href="#"><img src="https://static.igem.org/mediawiki/2015/b/ba/FAFU-CHINA_Dunk0.png" alt=""></a><span><font color="#FFFFFF">Fuzhou Dunk Internet Technology Corporation</font></span>
  
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<h2>Parts</h2>
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      <a href="#"><img src="https://static.igem.org/mediawiki/2015/f/f3/FAFU-CHINA_Fanshu_park0.png" alt=""></a><span><font color="#FFFFFF">FUNSHU PARK</font></span>
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<p>Length</p>
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<li>BBa_K1556000</li>
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<p>coding</p>
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<p>Mouse TdT</p>
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<p>1590</p>
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<small style="font-size: 8px">This part was submitted late, and is not eligible for medals and awards.</small>
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<p>Kenny Kostenbader</p>
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<p>Chem Eng</p>
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<img src="https://static.igem.org/mediawiki/2014/8/83/Rutgers_SL.png" />
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<p>Scott Lazaro</p>
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<p>Cell Bio &amp; Neurosci</p>
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<img src="https://static.igem.org/mediawiki/2014/4/42/Rutgers_WW.png" />
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<p>Wilson Wong</p>
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<img src="https://static.igem.org/mediawiki/2014/1/13/Rutgers_JP.png" />
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<p>Jay Patel</p>
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      <a href="#"><img src="https://static.igem.org/mediawiki/2015/b/bb/FAFU-CHINA_school_logo.png" alt=""></a>
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    <font color="#FFFFFF">Fujian Agriculture and Forestry University</font>
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<p>Sagar Khare</p>
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<p>Andrew Laudisi</p>
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<p>Lab Manager</p>
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<img class="down_arrow" src="https://static.igem.org/mediawiki/2014/f/f5/Rutgers_down.png" />
 
  
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<img src="https://static.igem.org/mediawiki/2014/5/5f/Rutgers_RJ.png" width="130" /> <p><strong>Dr Jones</strong> is a professor in the Rutgers Chemistry Department who researches modified nucleotides. He gave valuable feedback on our initial project ideas, and suggested a way to create (and characterize) 3'-acetylated thymidine triphosphate in our lab. We attempted this synthesis (involving pyridine and acetic anhydride) and got promising results (via LC/MS), but then we found out that TriLink (below) could simply synthesize and purify it for us for free.</p>
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<img src="https://static.igem.org/mediawiki/2014/f/fc/Rutgers_AN.png" width="130" /> <p><strong>Arun Nayar</strong> was on last year's Rutgers iGEM team, so he helped train us with various lab protocols.</p>
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<p>All of the pictured results on the Project page represent assays that were designed jointly by the students and Dr Khare, and carried out completely by the undergraduate team members in the Khare lab. Additional help was provided by other Rutgers students as well, namely: Diego Barreto, Wesley Okwemba, Neil Patel, Harsh Patel, and Samantha Ashley. The fluorescent gels were imaged using the "BioRad Gel Doc" machine in the Kalodimos lab (next door). Kenny did the web design and coding.</p>
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<img src="https://static.igem.org/mediawiki/2014/4/4b/Rutgers_Trilink.jpg" width="200" /> <p><strong>Trilink Biotech</strong> supported the project by custom-synthesizing acetylated thymidine triphosphate, and supplying it free of charge. Thanks TiLink!</p>
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<img src="https://static.igem.org/mediawiki/2014/e/e9/Rutgers_NEB.jpeg" width="200" /> <p><strong>NEB Inc</strong> supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge. Thanks NEB!</p>
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<img src="https://static.igem.org/mediawiki/2014/f/f7/Rutgers_Gen9.png" width="200" /> <p><strong>Gen9</strong> supported the project with a monetary donation. Thanks Gen9!</p>
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<section id="notebook-section">
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<h2>Protocols</h2>
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    wow = new WOW(
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<p>Click to download a copy of each protocol that we followed and/or wrote this summer:</p>
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<li><a href="https://static.igem.org/mediawiki/2014/2/23/Rutgers_a2.pdf">Testing the extent of hydrolysis of incorporated acetylated thymidines</a></li>
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+
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<li>In <strong>May</strong> and <strong>June</strong> we brainstormed and investigated ways to create acetylated thymidine in lab. After much much research into the matter, we decided it would require far to many resources to synthesize acetylated nucleoside monomers (without phosphates present) followed by difficult and expensive triphosphorylation (the stepwise installation of the triphosphate moiety). It would be far too expensive. Instead, we tried Dr Jones' suggestion of mixing acetic anhydride with thymidine triphosphate in pyridine, and this seemed to work (there was a peak at the right molecular weight on our LC/MS run :). This phase of the project ended when we discovered that TriLink could custom-synthesize acetylated thymidine triphosphate, and they would do it for free :)</li>
+
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<li>In <strong>July</strong> we expressed TdT in E. coli using the trusty pET-29b+ vector. Expression level was low (but still something!), so we were relieved when we found out that NEB would be happy to supply us with free a free TdT enzyme kit.</li>
+
var $anchor = $(this);
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<li>In <strong>August</strong> we ordered fluorescent primers to begin assaying with. It took the full month to test different methods for every step of the designed assay (especially fluorescence visualization) until we finally settled on the protocol(s) that we've uploaded (above)</li>
+
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<li><strong>September</strong> and <strong>October</strong> saw the bulk of our assaying. We had, however, neglected to move the mouse TdT gene from our pET-29b+ plasmid over to the standard iGEM pSB1C3 plasmid until early October. Our first Gibson Assembly did not work well (transformation yielded no colonies), so we ended up submitting our part late. That was a bummer.</li>
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<li>All lab members underwent Biosafety Level 2 training with the Rutgers Environmental Health &amp; Safety department.</li>
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<li>Public health and safety would be under little conceivable risk in the event of an accidental release of our biobrick part into the wild (in the form DNA or a transformed colony of cells), because the TdT enzyme expression is very low, and the enzyme is only capable of adding deoxynucleoside triphosphates to the 3' ends of DNA strands. Given that very little TdT would be produced, and that free 3' DNA ends are rare in living systems, we believe that the risk posed by our biobrick part is minimal.</li>
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Latest revision as of 03:45, 19 September 2015

Homepage

    Project

Design

Meeting

Notebook

Protocol

Result

Theory

Safety & Part

safety

Bio-safety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health. These prevention mechanisms include conduction of regular reviews of the bio-safety in laboratory settings, as well as strict guidelines to follow.


MORE

Organisms we used :

Without everyone's participation,our igem team would still be a plan....

Team

Xiaolei Huang

Instructor

Zujian Wu

Advisor

Taiyun Wei

Advisor

Bingfeng Zhou

Advisor

Penny

Advisor

Wenxu Wu

Web Advisor

Mucheng Cai

Dry Lab

Ruicheng Dai

Captain

Changlong Lu

Experiment Designer

Junhao Lu

Vice-Captain

Zhili Lai

Lecturer

LIANGDI CHEN

Web Designer

Tengzhou Huang

Dry Lab

Zirui Cheng

Dry Lab

Yifan Tang

Dry Lab

Tong Tong

Data Analyst

Human practice

Description:

To remove the barrier between science and normal people’s life, It is our responsibility to lead people into the wonderland of science. This is a lofty ideal, and thousands of people in many fields have already been working for it for years. But this time, we need some innovations, perhaps some funs too.
Play is not only a simple process. We believe that play is actually a way of being, a way of learning, a way of doing science. So now If u ever watched the TED of Beau Lotto . You’ll know that play provides everything you need in oder to be a scientist, and that is what motivates us. If we add rules to play, we have a game which is actually what project is. And who can be better than children to play? Then we tried to spread the idea that everyone has the potential to be scientist as long as being offered an opportunity. Instead of giving a dull lesson about biology, this year FAFU-China decided to teach children how to play correctly, how to play with principles from science, and finally they will find their own way to science....
Contact us:
Email:tripletau@126.com
Zipcode:350000
Fuzhou Dunk Internet Technology Corporation FUNSHU PARK Fujian Agriculture and Forestry University