Difference between revisions of "Team:Sheffield/Parts"

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{{Sheffield}}
 
 
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<title>A template page</title>
  
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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#shield  p {
<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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@media (min-width:768px) and (max-width:991px) {
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#shield  p {
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#shield li {
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      #shield span {
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      font-size:18px;
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</style>
  
  
<div class="column half_size">
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</head>
<div class="highlight">
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<h5>Note</h5>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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</div>
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</div>
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<body style="min-height:2000px;background-color:black">
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<nav class="navbar navbar-default navbar-fixed-top">
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      <div class="container">
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        <div class="navbar-header">
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          <div class="navbar-toggle collapsed" data-toggle="collapse" data-target="#navbar" aria-expanded="false" aria-controls="navbar">
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            <img src="https://static.igem.org/mediawiki/2016/a/a5/T--Sheffield--Website-home-mobile.png">
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          </div>
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          <!-- <a class="navbar-brand" href="#">Igem Sheff 16</a> -->
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        </div>
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        <div id="navbar" class="navbar-collapse collapse">
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          <ul class="nav navbar-nav">
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<li id="navimage" >
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<table id="imgtable">
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<th>
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<div class="navimagediv">
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<a href="https://2016.igem.org/Team:Sheffield"><img src="https://static.igem.org/mediawiki/2016/1/18/T--Sheffield--navbar-logo.png"></a>
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</div>
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</th>
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</table>
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            </li>
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    <li class="dropdown" id="li-home">
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              <a href="https://2016.igem.org/Team:Sheffield" >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-intro">
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<tr>
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<td>
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<p>HOME</p>
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</td>
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</tr>
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</table>
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  </center>
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              </a>
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            </li> 
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            <li class="dropdown">
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            <a href="https://2016.igem.org/Team:Sheffield/Description" class="dropdown-toggle"  >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-intro">
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<img src="https://static.igem.org/mediawiki/2016/5/53/T--Sheffield--Intro-Icon.png">
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</div>
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</center>
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</th>
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<tr>
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<td>
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<p>INTRO</p>
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</td>
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</tr>
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</table>
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  </center>
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              </a>
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            </li>         
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            <li class="dropdown">
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              <a href="https://2016.igem.org/Team:Sheffield/project" class="dropdown-toggle"  >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-project">
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<img src="https://static.igem.org/mediawiki/2016/8/86/T--Sheffield--icon-project.png">
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</div>
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</center>
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</th>
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<tr>
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<td>
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<p>PROJECT</p>
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</td>
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</tr>
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</table>
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  </center>
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              </a>
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            </li> 
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<li class="dropdown">
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              <a href="https://2016.igem.org/Team:Sheffield/Team" class="dropdown-toggle" >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-team">
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<img src="https://static.igem.org/mediawiki/2016/0/07/T--Sheffield--icon-team.png">
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</div>
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</center>
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</th>
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<tr>
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<td>
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<p>TEAM</p>
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</td>
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</tr>
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</table>
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  </center>
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              </a>
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            </li> 
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<li class="dropdown">
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              <a href="https://2016.igem.org/Team:Sheffield/Notebook" class="dropdown-toggle" >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-notebook">
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<img src="https://static.igem.org/mediawiki/2016/c/ce/T--Sheffield--icon-notebook.png">
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</div>
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</center>
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</th>
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<tr>
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<td>
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<p>NOTES</p>
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</td>
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</tr>
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</table>
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  </center>
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            <li class="dropdown">
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              <a href="https://2016.igem.org/Team:Sheffield/judging" class="dropdown-toggle" >
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  <center>
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<table id="navtable">
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<th>
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<center>
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<div id="nav-judging">
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<img src="https://static.igem.org/mediawiki/2016/3/35/T--Sheffield--icon-judging.png">
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</div>
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</center>
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</th>
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<tr>
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<td>
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<p>JUDGING</p>
 +
</td>
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</tr>
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</table>
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  </center>
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              </a>
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            </li> 
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        </div><!--/.nav-collapse -->
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      </div>
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    </nav>
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    <div id="shield">
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    <div class = "container" >
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<div class="spacer"></div>
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<div class="heading">
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<table>
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<th class="banner-img">
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<img src="https://static.igem.org/mediawiki/2016/6/67/T--Sheffield--heading-banner.png">
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</th>
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<th>
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<div class="heading-title"><h1 style="padding:0px">BIOBRICKS</h1></div>
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</th>
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<th class="banner-img">
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<img src="https://static.igem.org/mediawiki/2016/b/b2/T--Sheffield--heading-banner-flip.png">
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</table>
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                <div class="jumbotron">
 +
<h2><center>Introduction</center></h2>
 +
<p>Following our project we submitted 4 biobricks to iGEM 2016 registry. 2 of the biobricks included different hemerythrins genes: <i>mc</i> and <i>td</i>; one was an <i>sfgfp</i> gene under control of a strong promoter with an additional regulatory region operated by RyhB; the last biobricks was a modified strong promoter followed by a ribosomal binding site. Promoter sequences were modified using the iGEM biobrick Part: <a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>. All submitted biobricks are summarised in the table below. Unfortunately we were unable to clone Dcr, medium promoter and Med-RyhB-GFP into our pSB1C3 plasmid, due to XbaI and SpeI restriction sites having the same sticky-ends, thus creating self-ligated plasmid on multiple occasions.
 +
<center>
 +
<img style="width:70%;border:4px solid black" src="https://static.igem.org/mediawiki/2016/0/02/T--Sheffield--table1.png"
 +
</center></p>
 +
                </div>
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                <center>
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<table id="bricks-table">
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                      <tr>
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                            <td>
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                                <div class="brick-cell cell1-1">
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                                <h1 class="brick-header">BBa K2016000</h1>
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                                </div>
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                            </td>
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                            <td>
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                                <div class="brick-cell cell1-2">
 +
                                <h1 class="brick-header">BBa K2016003</h1>
 +
                                </div>
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                            </td>
 +
                      </tr>
 +
                      <tr>
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                            <td>
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                                <div class="brick-cell cell2-1">
 +
                                <h1 class="brick-header">BBa K2016004</h1>
 +
                                </div>
 +
                            </td>
 +
                            <td>
 +
                                <div class="brick-cell cell2-2">
 +
                                <h1 class="brick-header">BBa K2016005</h1>
 +
                                </div>
 +
                            </td>
 +
                      </tr>
 +
                </table>
 +
                </center>
 +
                <div class = "box0">
 +
                <center>
 +
                  <img src="https://static.igem.org/mediawiki/2016/a/a3/T--Sheffield--slider-intro.png">
 +
                <center>
 +
                </div>
 +
                <div class="box1">
 +
                    <div class="jumbotron">
 +
<h2><center>Strong constitutive promoter</center></h2>
 +
<p>During our project we have chosen 2 biobrick promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a> and <a href="http://parts.igem.org/Part:BBa_J23106">J23106</a>) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (<a href="http://parts.igem.org/Part:BBa_K2016000">BBa_K2016000</a>). </p>
 +
<center>
 +
<img style="width:40%" src="https://static.igem.org/mediawiki/2016/5/56/StrP.png">
 +
</center>
 +
<p><small><b>Figure 1: Map of BBa_K2016000 biobrick cloned into pSC1C3 plasmid.</b> Biobrick is cloned between XbaI and SpeI sites</small></p>
 +
<h1>Characterisation</h1>
 +
<p>We characterised our promoters by placing our sfGFP construct under the control of these promoters, and measuring their fluorescence as an indicator of gene expression.</p>
 +
<center>
 +
<img style="width:100%" src="https://static.igem.org/mediawiki/2016/5/59/T--Sheffield--RyhB-graph1.png">
 +
</center>
 +
<p><small><b>Figure 2. Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium (MedGFP) or strong promoters (StrGFP).</b></small></p><br/>
 +
                </div>
 +
                </div>
  
 +
                <div class="box2">
 +
                    <div class="jumbotron">
 +
<h2><center>McHr - hemerythrin</center></h2>
 +
<p><i>Methylococcus capsulatus</i> hemerythrin (McHr) is a protein expressed by methane-oxidising bacterium: <i>Methylococcus capsulatus</i>. The family of hemerythrins can undergo a slow process of oxygenation upon binding of iron which causes their colour change from colourless into red/yellow. Its mechanism of oxygenation has been discovered to be much more rapid than other hemerythrins we investigated (DcrHr or TdHr). This gene was synthesised based on the GeneBank sequence entry and cloned into the pSB1C3 plasmid.</p>
 +
<center>
 +
<img style="width:40%" src="https://static.igem.org/mediawiki/2016/c/ce/Mc.png">
 +
</center>
 +
<p><small><b>Figure 1: Map of BBa_K2016003 biobrick cloned into pSC1C3 plasmid.</b> Biobrick is cloned between XbaI and SpeI sites, however it is cloned in reverse orientation due to XbaI and SpeI enzymes having the same overhangs after restriction digest. To use this biobrick future teams should use PCR apmlification rather than restriction digest to clone this biobrick.</small></p>
 +
<h1>Characterisation</h1>
 +
<p>We believe we were able to express McHr in <i>E.coli</i> cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter, however its induction is not as clear as in the other bands indicating that it may need further characterisation (Fig. 2).</p>
 +
<center>
 +
<img style="width:70%" src="https://static.igem.org/mediawiki/2016/8/80/T--Sheffield--Magda4a_aa.png">
 +
</center>
 +
                <p><small><b>Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.</b>For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Mc (14.7 kDa) we can see induction of expression is less clear after adding IPTG, and should be investigated further.</small></p><br/>
 +
                </div>
 +
                </div>
  
 +
                <div class="box3">
 +
                    <div class="jumbotron">
 +
<h2><center>TdHr - hemerythrin</center></h2>
 +
<p>TdHr is a protein expressed by <i>Themiste dyscritum</i>, a eukaryotic marine organism, in which this protein functions as an iron-binding transporter, much like our own haemoglobin. Td undergoes a slow process of oxygenation upon binding of iron and gains a violet/pink colour. This gene was codon-optimised for the use in <i>E. coli</i> and cloned into the pSB1C3 plasmid.</p>
 +
<center>
 +
<img style="width:40%" src="https://static.igem.org/mediawiki/2016/7/7e/Td.png">
 +
</center>
 +
<p><small><b>Figure 1: Map of BBa_K2016004 biobrick cloned into pSC1C3 plasmid.</b> Biobrick is cloned between XbaI and SpeI sites.</small></p>
 +
<h1>Characterisation</h1>
 +
<p>We were able to express TdHr in <i>E.coli</i> cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter (Fig. 2).</p>
 +
<center>
 +
<img style="width:70%" src="https://static.igem.org/mediawiki/2016/8/80/T--Sheffield--Magda4a_aa.png">
 +
</center>
 +
                <p><small><b>Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.</b>For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Td (13.5 kDa) we can see clear induction of expression after adding IPTG.</small></p><br/>
  
<div class="column half_size">
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                </div>
 
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                </div>
<h5>Adding parts to the registry</h5>
+
<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
+
<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
+
</div>
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<div class="column half_size">
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<h5>What information do I need to start putting my parts on the Registry?</h5>
+
<p>The information needed to initially create a part on the Registry is:</p>
+
<ul>
+
<li>Part Name</li>
+
<li>Part type</li>
+
<li>Creator</li>
+
<li>Sequence</li>
+
<li>Short Description (60 characters on what the DNA does)</li>
+
<li>Long Description (Longer description of what the DNA does)</li>
+
<li>Design considerations</li>
+
</ul>
+
 
+
<p>
+
We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
+
 
+
</div>
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<div class="column half_size">
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<h5>Inspiration</h5>
+
<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h5>Part Table </h5>
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<groupparts>iGEM2016 Example</groupparts>
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<h2><center>Str-RyhB-GFP - Superfolder GFP under the control of RyhB</center></h2>
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<p>Our superfolder GFP was cloned to be operated by RyhB on the RNA interference level. RyhB is responsive to iron concentrations within the cell and therefore provides us with a viable tool needed for iron detection. Strong or medium promoters were used for expression of gfp on different levels. However, due to difficulties with cloning, only the GFP under control of strong promoter was submitted to the iGEM registry. The gene was cloned into pSB1C3 vector as shown below.</p>
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<p><small><b>Figure 1: Map of BBa_K2016005 biobrick cloned into pSC1C3 plasmid.</b> Biobrick is cloned between XbaI and SpeI sites.</small></p>
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<h1>Characterisation</h1>
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<p>We demonstrated that our RhyB reporter system responds to increased iron concentrations by adding iron to our strains that had been transformed with our Str-RyhB-GFP and Med-RyhB-GFP constructs, after which we detected a significant increase in fluorescence</p> 
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                <p><small><b>Figure 2. Change in GFP expression after 90 minutes following addition of FeCl<sub>3</sub> to a final concentration of 100 μM (A) or without addition of FeCl<sub>3</sub> (B)</b>. There is a significant increase in fluorescence upon addition of iron over the course of 90 minutes (A), however this was not seen over the same time period without addition of iron (B). Significance was assessed using a paired t-test and is conveyed with * <0.05, **<0.01, *** <0.001, **** <0.0001. </small></p><br/>
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Latest revision as of 14:40, 19 October 2016

A template page

BIOBRICKS

Introduction

Following our project we submitted 4 biobricks to iGEM 2016 registry. 2 of the biobricks included different hemerythrins genes: mc and td; one was an sfgfp gene under control of a strong promoter with an additional regulatory region operated by RyhB; the last biobricks was a modified strong promoter followed by a ribosomal binding site. Promoter sequences were modified using the iGEM biobrick Part: BBa_J23100. All submitted biobricks are summarised in the table below. Unfortunately we were unable to clone Dcr, medium promoter and Med-RyhB-GFP into our pSB1C3 plasmid, due to XbaI and SpeI restriction sites having the same sticky-ends, thus creating self-ligated plasmid on multiple occasions.

BBa K2016000

BBa K2016003

BBa K2016004

BBa K2016005

Strong constitutive promoter

During our project we have chosen 2 biobrick promoters (J23100 and J23106) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (BBa_K2016000).

Figure 1: Map of BBa_K2016000 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites

Characterisation

We characterised our promoters by placing our sfGFP construct under the control of these promoters, and measuring their fluorescence as an indicator of gene expression.

Figure 2. Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium (MedGFP) or strong promoters (StrGFP).


McHr - hemerythrin

Methylococcus capsulatus hemerythrin (McHr) is a protein expressed by methane-oxidising bacterium: Methylococcus capsulatus. The family of hemerythrins can undergo a slow process of oxygenation upon binding of iron which causes their colour change from colourless into red/yellow. Its mechanism of oxygenation has been discovered to be much more rapid than other hemerythrins we investigated (DcrHr or TdHr). This gene was synthesised based on the GeneBank sequence entry and cloned into the pSB1C3 plasmid.

Figure 1: Map of BBa_K2016003 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites, however it is cloned in reverse orientation due to XbaI and SpeI enzymes having the same overhangs after restriction digest. To use this biobrick future teams should use PCR apmlification rather than restriction digest to clone this biobrick.

Characterisation

We believe we were able to express McHr in E.coli cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter, however its induction is not as clear as in the other bands indicating that it may need further characterisation (Fig. 2).

Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Mc (14.7 kDa) we can see induction of expression is less clear after adding IPTG, and should be investigated further.


TdHr - hemerythrin

TdHr is a protein expressed by Themiste dyscritum, a eukaryotic marine organism, in which this protein functions as an iron-binding transporter, much like our own haemoglobin. Td undergoes a slow process of oxygenation upon binding of iron and gains a violet/pink colour. This gene was codon-optimised for the use in E. coli and cloned into the pSB1C3 plasmid.

Figure 1: Map of BBa_K2016004 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.

Characterisation

We were able to express TdHr in E.coli cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter (Fig. 2).

Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Td (13.5 kDa) we can see clear induction of expression after adding IPTG.


Str-RyhB-GFP - Superfolder GFP under the control of RyhB

Our superfolder GFP was cloned to be operated by RyhB on the RNA interference level. RyhB is responsive to iron concentrations within the cell and therefore provides us with a viable tool needed for iron detection. Strong or medium promoters were used for expression of gfp on different levels. However, due to difficulties with cloning, only the GFP under control of strong promoter was submitted to the iGEM registry. The gene was cloned into pSB1C3 vector as shown below.

Figure 1: Map of BBa_K2016005 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.

Characterisation

We demonstrated that our RhyB reporter system responds to increased iron concentrations by adding iron to our strains that had been transformed with our Str-RyhB-GFP and Med-RyhB-GFP constructs, after which we detected a significant increase in fluorescence

Figure 2. Change in GFP expression after 90 minutes following addition of FeCl3 to a final concentration of 100 μM (A) or without addition of FeCl3 (B). There is a significant increase in fluorescence upon addition of iron over the course of 90 minutes (A), however this was not seen over the same time period without addition of iron (B). Significance was assessed using a paired t-test and is conveyed with * <0.05, **<0.01, *** <0.001, **** <0.0001.