Difference between revisions of "Team:Aix-Marseille/Design"

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{{Aix-Marseille}}
 
 
<html>
 
<html>
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<title>Chew fight</title>
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<meta name="viewport" content="width=device-width, initial-scale=1.0">
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<meta http-equiv="content-type" content="text/html; charset=UTF-8">
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        <link rel="stylesheet" href="https://2015.igem.org/Team:NRP-UEA-Norwich/bootstrapcss?action=raw&amp;ctype=text/css" type="text/css">
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<link rel="stylesheet" href="https://2015.igem.org/Team:NRP-UEA-Norwich/mobilecss?action=raw&amp;ctype=text/css" type="text/css">
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        <script type="text/javascript" src="https://2015.igem.org/Team:NRP-UEA-Norwich/bootstrap.js?action=raw&ctype=text/javascript"></script>
  
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<h2>Design</h2>
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        <script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE7.js"></script>
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<p>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page. If you are going for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Applied Design award</a>, you should also complete this page and tell us what you did.
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</p>
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        <![endif]-->
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        <script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE9.js"></script>
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<style type="text/css">
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.affix {
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background-color: #8E3B8C;
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}
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footer {
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padding: 10px 0px 10px 0px;
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}
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header {
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background-color: #8E3B8C;
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padding: 0px;
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}
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#nav >li > a {
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color: #FFFFFF !important;
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} .bg-1 {
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background: url(https://static.igem.org/mediawiki/2015/f/f3/FrsJhjG.jpg);background-repeat:no-repeat;
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min-height: 600px;
  
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<div class="highlightBox">
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}
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<h4>Note</h4>
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.head-1 {
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best Applied Design award</a> and/or the <a href="https://2015.igem.org/Judging/Awards#Medals">functional prototype gold medal criterion</a>, you must fill out this page.</p>
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text-shadow: 0px 0px 10px rgba(150, 150, 150, 1), 0px 0px 10px rgba(150, 150, 150, 1), 0px 0px 10px rgba(150, 150, 150, 1), 0px 0px 10px rgba(150, 150, 150, 1);
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</div>
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}
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</style>
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<link rel="stylesheet" href="https://cdnjs.cloudflare.com/ajax/libs/animate.css/3.4.0/animate.min.css">
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<script src="https://cdnjs.cloudflare.com/ajax/libs/wow/1.1.2/wow.min.js"></script>
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              <script>
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              new WOW().init();
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              </script>
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</head>
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<body>
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<header class="header" >
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        <div class="container">
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            <div class="row">
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                <nav class="navbar navbar-default " role="navigation" data-spy="affix" data-offset-top="600">
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                  <div class="navbar-inner"> 
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                    <div class="navbar-header">
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                        <button type="button" class="navbar-toggle" data-toggle="collapse" data-target="#bs-example-navbar-collapse-1">
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                            <span class="sr-only">Toggle navigation</span>
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                            <span class="icon-bar"></span>
  
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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                        </button>
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                        <a href="/Team:Aix Marseille" class="navbar-brand hidden-lg hidden-md hidden-sm" ><https://static.igem.org/mediawiki/2015/6/6e/OHSATfM.png" class="img-responsive" width="80" height="80"></a>
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                    </div>
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                                <li><a href="/Team:Aix-Marseille"  class="logo"><img src="https://static.igem.org/mediawiki/2015/6/6e/OHSATfM.png" class="img-responsive" width="85" height="85"></a></li>
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                                    <li  ><a href="/Team:Aix-Marseille">Home</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Project/Description">Overview</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Design">Project design</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Parts">Parts</a></li>                                 
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                                      <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Team</a>
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                                        <li ><a href="/Team:Aix-Marseille/Team">Members</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Attributions">Attributions</a></li>                                 
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                                      <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Notebook</a>
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                                        <li ><a href="/Team:Aix-Marseille/Notebook">Calendar</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Results">Results</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Protocols">Protocols</a></li>                                 
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                                      </ul>
  
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<p>
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                                    <li  ><a href="/Team:Aix-Marseille/Practices">Practices</a></li> 
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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</p>
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                                    <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle" data-toggle="dropdown">Interlab Study</a>
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                                      <ul class="dropdown-menu">
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                                        <li ><a href="/Team:Aix-Marseille/Notebook2">Calendar</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Measurement">Results</a></li>
 +
                                        <li ><a href="/Team:Aix-Marseille/Protocols2">Protocols</a></li>
 +
                                        <li ><a href="/Team:Aix-Marseille/Sequencing/data">Sequencing data</a></li>                                 
 +
                                      </ul>
 +
                                    <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle" data-toggle="dropdown">Modeling</a>
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                                      <ul class="dropdown-menu">
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                                        <li ><a href="/Team:Aix-Marseille/Modeling">Modeling</a></li>
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                                        <li ><a href="/Team:Aix-Marseille/Software">Software</a></li>                                 
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                                      </ul>   
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                      <li class="dropdown " id="dropdown"><a href="/Team:Aix-Marseille/Achievement" class="dropdown-toggle"  data-toggle="dropdown">Achievement</a></li>
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                                  </li>
  
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</div>
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                                </ul>
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                          </div>
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                </div>       
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                </nav>
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            </div>
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        </div>
  
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    </header>
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 +
    <!-- end hearo section -->
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    <div class="clearfix"></div>
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      <!-- start section 1 -->
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    <section style="padding:30px 0px 50px;" class="arrow_box" id="team">
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 +
    <div class="container">
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    <div class="row">
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    <div class="col-md-6 left">
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    <h2 class="title wow bounce in up"><span style="color:#8E3B8C"><span style="font-family:Armalite Rifle">Project design</h2></span></span>
 +
    <div class="space30"></div>
 +
    <p class="space20"><div align="justify"><span style="font-family: Courier New">Our project this year is to work on the <b>chewing gum degradation</b>.
 +
Our research on this topic reveals that chewing gum is composed of different compounds. They are hydrophilic or hydrophobic. When you chew a chewing gum, the hydrophilic part is solubilized by your saliva. Once done, you throw away the hydrophobic part called the gum base. The gum base is composed of polymers : polyisoprene and butadiene. </div></p></span>
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 +
<p class="space100"><div align="justify"><span style="font-family: Courier New">So we searched for enzymes which are able to degrade polymers. We found two enzymes: <b>lipoxygenases and laccases</b>.</div></p></span>
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 +
<p class="space20"><div align="justify"><span style="font-family: Courier New">At the beginning, our main problem was that laccase is able to oxidize the X component. But we did not want to use it in our project because of its scarcity, its toxicity and impact on the environment. We found that the cytochrome C which uses iron could replace it and be oxidized by laccases.
 +
We decided to try and asked if it could help to degrade our polymers. We selected several enzymes that we believed could be great to use in <i>E.coli</i> strain.</div></p></span>
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 +
    </div>
 +
    <div class="col-md-5 col-md-offset-1  col-sm-offset-1  space30">
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                            <img src="https://static.igem.org/mediawiki/2015/7/7b/Histo.jpg" class="img-responsive" width="450" height="300">
 +
                          <div align="center"><img src="https://static.igem.org/mediawiki/2015/9/95/Lac.png" class="img-responsive" width="400" height="400"></div>
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                        </div>
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                    </div>
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    </div>
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            <div class="space30"></div>
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    </section>
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    <div class="clearfix"></div>
 +
      <!-- start section 2 -->
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<section style="padding:30px 0px 30px;background-color: #FBF1FB;" "background:#fbf1fb;"="" class="bg-2 arrow_box2;">   
 +
    <div style ="background:#FBF1FB;">
 +
    <div class="container">
 +
    <div class="row">
 +
 +
    <div class="col-md-6 left">
 +
    <h2 class="title wow bounce in up"><span style ="color:#000000"><span style="font-family:Armalite Rifle">Our strategy</span></span></h2>
 +
    <div class="space30"></div>
 +
    <p class="space20"><div align="justify"><span style="font-family:Courier New"><span style ="color:#000000">Our strategy is to use a reduced laccase which will be oxidized by the oxygen present in the air.</p></span></span>
 +
<p><span style="font-family:Courier New"><span style ="color:#000000">To return at a reduced state, laccase will oxidize the cytochrom C previously excited by light.</p></span></span>
 +
<p><span style="font-family:Courier New"><span style ="color:#000000">When the cytochrome C will come back to its reduced state, the polymers will be in a radical state.</p></span></span>
 +
<p><span style="font-family:Courier New"><span style ="color:#000000">Thanks to oxygen, it will form an epoxide that will be hydrolyzed and become a diol.</p></span></span>
 +
<p><span style="font-family:Courier New"><span style ="color:#000000">So we think that the polymer degradation will be more efficient by this method.
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</p></span></div> </span>
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 +
 +
    </div>
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    <div class="col-md-5 col-md-offset-1  col-sm-offset-1  space30">
 +
                 
 +
                            <img src="https://static.igem.org/mediawiki/2015/9/96/Laccase_reaction.png" class="img-responsive" width="450" height="450">
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 +
                        </div>
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                        </div>
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                    </div>
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 +
    </div>
 +
</section>
 +
<div class="clearfix"></div> 
 +
 +
  <!--start section 3-->
 +
<section style="padding:50px 0px 30px;" class="arrow_box" id="team">
 +
   
 +
    <div class="container">
 +
    <div class="row">
 +
    <div class="col-md-6 left">
 +
    <h2 class="title wow bounce in up"><span style="color:#8E3B8C"><span style="font-family:Armalite Rifle">Our constructions</h2></span></span>
 +
    <div class="space30"></div>
 +
    <p class="space20"><div align="justify"><span style="font-family: Courier New">First, we will produce our enzymes independantly (lippoxygenase, laccase and cytochrome C). Usually, cytochrome C uses the Sec-pathway and folds into the periplasm. Howerver, we could not produce proteins that help the periplasmic folding. So we added CXXCH and CCHL sequences that will help the cytochrome C to use the Tat-pathway and allowed its cytoplasmic folding. Then we will test all the enzymes and different combinations on polymers.
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</div></p></span>
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<p class="space10"><div align="justify"><span style="font-family: Courier New">To test our enzymes, we will mesure the oxygen level with an oxymeter and by spectrophotometry.</div></p></span>
 +
 +
<p class="space50"><div align="justify"><span style="font-family: Courier New">We will select enzymes with the best results: one lipoxygenase, one laccase and one cytochrome C.
 +
Using a linker, we will fuse the selected laccase with the selected cytochrome C and test again the activity. We will compared our results. </div></p></span>
 +
 +
 +
    </div>
 +
    <div class="col-md-5 col-md-offset-1  col-sm-offset-1  space100">
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                            <img src="https://static.igem.org/mediawiki/2015/d/de/LKKEx94.png" class="img-responsive">
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<img src="https://static.igem.org/mediawiki/2015/c/c8/Cons2.png" class="img-responsive" "space100">
 +
                         
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                        </div>
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                    </div>
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 +
    </div>
 +
            <div class="space30"></div>
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    </section>
 +
  <div class="clearfix"></div> 
 +
   
 +
<!--start section 4-->
 +
 +
<section style="padding:30px 0px 30px;background-color: #FBF1FB;" "background:#fbf1fb;"="" class="bg-2 arrow_box2;">   
 +
    <div style ="background:#FBF1FB;">
 +
    <div class="container">
 +
    <div class="row">
 +
 +
    <div class="col-md-6 left">
 +
    <h2 class="title wow bounce in up"><span style ="color:#000000"><span style="font-family:Armalite Rifle">Production</span></span></h2>
 +
    <div class="space30"></div>
 +
    <p><div align="justify"><span style ="color:#000000"><span style="font-family:Courier New">Once our constructions done, we transform its into BL21 cells.
 +
These <i>E.coli</i> K-12 allow the expression of proteins controlled by the T7 promotor and induced by IPTG.
 +
Bacteria cells were resuspended into a lysis buffer and were sonicated. Sonication of bacteria is used to break cells that contain our proteins to be purified. After, a centrifugation separates broken/unbroken cells and allows us to collect soluble proteins into the supernatant.
 +
</p></span></div> </span>
 +
 +
 +
    </div>
 +
    <div class="col-md-5 col-md-offset-1  col-sm-offset-1  space30">
 +
                 
 +
                            <img src="https://static.igem.org/mediawiki/2015/9/90/Culturecells.jpg" class="img-responsive" width="450" height="450">
 +
 
 +
                        </div>
 +
                        </div>
 +
                    </div>
 +
   
 +
    </div>
 +
    <div class="space30">
 +
    </div>
 +
</section>
 +
<div class="clearfix"></div>
 +
 +
<!--start section 5-->
 +
<section style="padding:30px 0px 50px;" class="arrow_box" id="team">
 +
   
 +
    <div class="container">
 +
    <div class="row">
 +
    <div class="col-md-6 left">
 +
    <h2 class="title wow bounce in up"><span style="color:#8E3B8C"><span style="font-family:Armalite Rifle">Purification</h2></span></span>
 +
    <div class="space30"></div>
 +
    <p class="space100"><div align="justify"><span style="font-family: Courier New">We added a His-tag following the sequence of genes coding our proteins.
 +
 +
Using columns containing nickel ions, His-tagged proteins are bound with high specificity and affinity. Other proteins pass through the matrix during the washing step. His-tagged proteins are eluted using imidazole buffer.
 +
</div></p></span>
 +
 +
 +
 +
    </div>
 +
    <div class="col-md-5 col-md-offset-1  col-sm-offset-1  space30">
 +
                 
 +
                            <img src="https://static.igem.org/mediawiki/2015/a/a1/Purif.jpg" class="img-responsive" width="450" height="300">
 +
 +
                       
 +
                        </div>
 +
                       
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                    </div>
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 +
    </div>
 +
            <div class="space30"></div>
 +
 +
    </section>
 +
<div class="clearfix"></div>
 +
      <!-- start social section -->
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    <section style="padding:30px 0px;" class="arrow_box" id="ethics">
 +
   
 +
    <div class="container">
 +
    <div class="row">
 +
    <div class="col-md-12 text-center">
 +
<h1 class="blue">MORE INFORMATION ON FACEBOOK !</h1>
 +
                    <ul class="list-inline space80 icon">
 +
                        <li><a href="https://www.facebook.com/iGEM.AMU?fref=ts"><img src="https://static.igem.org/mediawiki/2015/e/ed/FacebookAMU.png" width="200" height="200"" class="img-grey mautomargin"></a></li>
 +
                    </ul>
 +
 +
                </div>
 +
    </div>
 +
    </div>
 +
 +
    </section>
 +
      <div class="clearfix"></div>
 +
      <!-- start sponsors section -->
 +
<section style="padding:50px 0px;" class="arrow_box-2">
 +
       
 +
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                        <p class="fsize18 space10">Chew figth project, for the iGEM competition. See you soon in Boston !</p>
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Latest revision as of 23:56, 18 September 2015

Chew fight

Project design

Our project this year is to work on the chewing gum degradation. Our research on this topic reveals that chewing gum is composed of different compounds. They are hydrophilic or hydrophobic. When you chew a chewing gum, the hydrophilic part is solubilized by your saliva. Once done, you throw away the hydrophobic part called the gum base. The gum base is composed of polymers : polyisoprene and butadiene.

So we searched for enzymes which are able to degrade polymers. We found two enzymes: lipoxygenases and laccases.

At the beginning, our main problem was that laccase is able to oxidize the X component. But we did not want to use it in our project because of its scarcity, its toxicity and impact on the environment. We found that the cytochrome C which uses iron could replace it and be oxidized by laccases. We decided to try and asked if it could help to degrade our polymers. We selected several enzymes that we believed could be great to use in E.coli strain.

Our strategy

Our strategy is to use a reduced laccase which will be oxidized by the oxygen present in the air.

To return at a reduced state, laccase will oxidize the cytochrom C previously excited by light.

When the cytochrome C will come back to its reduced state, the polymers will be in a radical state.

Thanks to oxygen, it will form an epoxide that will be hydrolyzed and become a diol.

So we think that the polymer degradation will be more efficient by this method.

Our constructions

First, we will produce our enzymes independantly (lippoxygenase, laccase and cytochrome C). Usually, cytochrome C uses the Sec-pathway and folds into the periplasm. Howerver, we could not produce proteins that help the periplasmic folding. So we added CXXCH and CCHL sequences that will help the cytochrome C to use the Tat-pathway and allowed its cytoplasmic folding. Then we will test all the enzymes and different combinations on polymers.

To test our enzymes, we will mesure the oxygen level with an oxymeter and by spectrophotometry.

We will select enzymes with the best results: one lipoxygenase, one laccase and one cytochrome C. Using a linker, we will fuse the selected laccase with the selected cytochrome C and test again the activity. We will compared our results.

Production

Once our constructions done, we transform its into BL21 cells. These E.coli K-12 allow the expression of proteins controlled by the T7 promotor and induced by IPTG. Bacteria cells were resuspended into a lysis buffer and were sonicated. Sonication of bacteria is used to break cells that contain our proteins to be purified. After, a centrifugation separates broken/unbroken cells and allows us to collect soluble proteins into the supernatant.

Purification

We added a His-tag following the sequence of genes coding our proteins. Using columns containing nickel ions, His-tagged proteins are bound with high specificity and affinity. Other proteins pass through the matrix during the washing step. His-tagged proteins are eluted using imidazole buffer.

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  • Aix Marseille Université
  • Marseille
  • France

Chew figth project, for the iGEM competition. See you soon in Boston !