Team:UFMG Brazil/Project/Parts

From 2014.igem.org

(Difference between revisions)
 
(9 intermediate revisions not shown)
Line 1: Line 1:
{{UFMG_Brazil/css}}
{{UFMG_Brazil/css}}
-
<html>
+
<html class=" desktop landscape" lang="en">
 +
 
<head>
<head>
-
<style>
 
-
#wrapper_parts{
 
-
 
-
padding: 2em;
 
-
background: #FFFFFF;
 
-
height: auto;
 
-
width: auto;
 
-
 
-
}
 
-
 
-
.parts_content{
 
-
text-align: justified;
 
-
padding: 1em;
 
-
}
 
-
</style>
 
-
 
     <meta http-equiv="content-type" content="text/html; charset=UTF-8">
     <meta http-equiv="content-type" content="text/html; charset=UTF-8">
-
     <title>Home</title>
+
     <title>Home UFMG Team</title>
     <meta charset="utf-8">
     <meta charset="utf-8">
     <meta name="format-detection" content="telephone=no">
     <meta name="format-detection" content="telephone=no">
-
     <link rel="stylesheet" href="https://dl.dropboxusercontent.com/u/80760623/igem2014/css/grid.css">
+
     <link rel="icon" href="images/favicon.ico" type="image/x-icon">
-
     <link rel="stylesheet" href="https://dl.dropboxusercontent.com/u/80760623/igem2014/css/style.css">
+
     <link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/grid.css&action=raw&ctype=text/css">
-
     <link rel="stylesheet" href="https://dl.dropboxusercontent.com/u/80760623/igem2014/css/font-awesome.css" >  
+
     <link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/style.css&action=raw&ctype=text/css">
-
     <link rel="stylesheet" href="https://dl.dropboxusercontent.com/u/80760623/igem2014/css/owl.carousel.css" >  
+
     <link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/font-awesome.css&action=raw&ctype=text/css" >  
-
     <link rel="stylesheet" href="https://fonts.googleapis.com/css?family=Roboto:300,400,500,700" >
+
     <link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/owl.carousel.css&action=raw&ctype=text/css" >  
-
    <link rel="stylesheet" href="https://fonts.googleapis.com/css?family=Bevan" >  
+
     <!--[if lt IE 9]>
     <!--[if lt IE 9]>
         <div style=' clear: both; text-align:center; position: relative;'>
         <div style=' clear: both; text-align:center; position: relative;'>
Line 34: Line 18:
             </a>
             </a>
         </div>
         </div>
-
         <script src="https://dl.dropboxusercontent.com/u/80760623/igem2014/js/html5shiv.js"></script>
+
         <script src="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/html5shiv.js&action=raw&ctype=text/javascript"></script>
-
         <link rel="stylesheet" type="text/css" media="screen" href="css/ie.css">
+
         <link rel="stylesheet" type="text/css" media="screen" href="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/ie.css&action=raw&ctype=text/css">
     <![endif]-->
     <![endif]-->
-
   
+
     <script>
-
     <script src="https://dl.dropboxusercontent.com/u/80760623/igem2014/js/jquery.js"></script>
+
      document.getElementById('top-section').style.height = '10px';
-
    <script src="https://dl.dropboxusercontent.com/u/80760623/igem2014/js/jquery-migrate-1.2.1.js"></script>
+
      var left_menu = document.querySelectorAll(".left-menu");
-
    <script src="https://dl.dropboxusercontent.com/u/80760623/igem2014/js/owl.carousel.js"></script>
+
      for (var i = 0, length = left_menu.length; i < length; i++) {
-
<script src="https://dl.dropboxusercontent.com/u/80760623/igem2014/js/script.js"></script>
+
        left_menu[i].style.display = 'none';
-
<script>
+
      }
-
$( document ).ready(function() {
+
      var right_menu = document.querySelectorAll(".right-menu");
-
    $('#igemmenulogo').click(function(){
+
      for (var i = 0, length = right_menu.length; i < length; i++) {
-
        toggleMenu();
+
        right_menu[i].style.display = 'none';
-
     });
+
      }
-
});
+
    </script>
-
function toggleMenu(){
+
    <style>
-
        if($('#top-section').css("height") == "25px"){
+
 
-
            $('#top-section').css("height", "10px");
+
.tg  {border-collapse:collapse;border-spacing:0;border-color:#ccc;}
-
            $('.left-menu').hide();
+
.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#fff;}
-
            $('.right-menu').hide();
+
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#f0f0f0;}
-
        }
+
.tg .tg-eyl8{font-weight:bold;background-color:#ecf4ff}
-
        else{
+
 
-
            $('#top-section').css("height", "25px");
+
 
-
            $('.left-menu').show();  
+
     .thumbnail {
-
            $('.right-menu').show();
+
      position: relative;
-
        }
+
      overflow: hidden;
-
}
+
      text-align: center;
-
toggleMenu();
+
      padding: 0 0 30px;
-
</script>
+
      border-bottom: 0px;
 +
      margin-bottom: 0px;
 +
      -webkit-transition: all 0.3s ease;
 +
      transition: all 0.3s ease;
 +
    }
 +
    .thumbnail:after {
 +
      position: absolute;
 +
      content: "";
 +
      background: none;
 +
      bottom: 0;
 +
      left: 45%;
 +
      height: 13px;
 +
      width: 25px;
 +
      -webkit-transition: all 0.3s ease;
 +
      transition: all 0.3s ease;
 +
    }
 +
    .thumbnail:hover:after {
 +
      background: none;
 +
    }
 +
    .thumbnail:hover {
 +
      border-bottom: none;
 +
    }
 +
    .header {
 +
      padding-bottom: 0px;
 +
    }
 +
    </style>
</head>
</head>
<body id="top">
<body id="top">
 +
   <!--========================================================
   <!--========================================================
                             HEADER  
                             HEADER  
   =========================================================-->
   =========================================================-->
    
    
-
<header id="header">
+
<header id="header" class="header">
     <div class="banner1">
     <div class="banner1">
       <div id="stuck_container">
       <div id="stuck_container">
Line 76: Line 86:
                       <div class="grid_12">
                       <div class="grid_12">
                             <ul class="sf-menu">
                             <ul class="sf-menu">
-
                                 <li class="current"><a href="https://2014.igem.org/Team:UFMG_Brazil">Home</a></li>
+
                                 <li><a href="https://2014.igem.org/Team:UFMG_Brazil">Home</a></li>
                                 <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Team">Team</a></li>
                                 <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Team">Team</a></li>
-
                                 <li><a href="#">Safety</a></li>
+
                                 <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Safety">Safety</a></li>
-
                                 <li><a href="#">Project</a>
+
                                 <li class="current"><a href="#">Project</a>
-
                                     <ul>
+
                                     <ul style="display:none;">
-
                                         <li><a href="#">Parts</a></li>
+
                                         <li><a href="https://2014.igem.org/Team:UFMG Brazil/Project/Intro">Introduction</a></li>
-
                                         <li><a href="#">Protocols</a></li>
+
                                        <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Project/Parts">Parts</a></li>
-
                                         <li><a href="#">Modelling</a></li>
+
                                         <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Project/Protocols">Protocols and results</a></li>
-
                                         <li><a href="#">Human Practices</a></li>
+
                                         <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Project/Modelling">Modelling</a></li>
-
                                         <li><a href="#">Notebook</a></li>
+
                                         <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Project/Policy">Policy and practices</a></li>
 +
                                         <li><a href="https://2014.igem.org/Team:UFMG_Brazil/Project/Notebook">Notebook</a></li>
                                     </ul>
                                     </ul>
                                 </li>
                                 </li>
-
                                 <li><a href="#">Sponsors</a></li>
+
                                 <li><a href="#">Thanks to</a>
-
                                 <li><div style="padding-top:5px;"><img id="igemmenulogo" src="https://dl.dropboxusercontent.com/u/80760623/igem2014/images/Igemlogo_300px.png" alt=""></div></li>
+
                                    <ul style="display:none;">
 +
                                      <li>
 +
                                        <a href="https://2014.igem.org/Team:UFMG_Brazil/sponsors">Sponsors</a>
 +
                                      </li>
 +
                                      <li>
 +
                                        <a href="https://2014.igem.org/Team:UFMG_Brazil/attributions">Attributions</a>
 +
                                      </li>
 +
                                    </ul>
 +
                                 </li>
 +
                                <li>
 +
                                  <div>
 +
                                    <div style="padding-top:5px; display:inline-block;"><a href="https://igem.org/"><img id="igemmenulogo" src="https://static.igem.org/mediawiki/2014/6/60/Igemlogo_300px.png" alt=""></a></div>
 +
                                    <div style="padding-top:24px; display:inline-block;"><a href="#"><img id="topmenutoggle" src="https://static.igem.org/mediawiki/2014/5/5b/Onoff.png" alt="" style="cursor:pointer"></a></div>
 +
                                  </div>
 +
                                </li>
                             </ul>
                             </ul>
                         </div>
                         </div>
Line 96: Line 121:
           </nav>
           </nav>
       </div>
       </div>
 +
       <div class="container">
       <div class="container">
           <div class="row">
           <div class="row">
               <div class="grid_12">
               <div class="grid_12">
                     <div class="unit" style="padding-top:50px; padding-bottom: 20px;">
                     <div class="unit" style="padding-top:50px; padding-bottom: 20px;">
-
                       <div class="title1">- Our Parts -</div>
+
                       <div class="title1">- parts -</div>
                     </div>
                     </div>
-
                  <!--<h1><a href="index.html"><img src="images/logo.png" alt=""></a></h1>-->
 
                   <div class="slogan">Check out our amazing design!</div>
                   <div class="slogan">Check out our amazing design!</div>
               </div>     
               </div>     
Line 111: Line 136:
<div class="clear"></div>
<div class="clear"></div>
-
<div id="wrapper_parts">
+
  <!--========================================================
 +
                            CONTENT
 +
  =========================================================-->
-
<div class="parts_content">
+
<div id="content">
 +
    <div class="bg4 p50" style="padding: 50px 0 31px;">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="grid_12">
 +
                  <h2>Our building blocks!</h2>
 +
                    <div class="block1">
-
<p>As a proxy for the detection of L-DNA, we decided to build first a generic DNA sensor
+
                      <!--<h5><a href="#" onclick="return false;">Whitout their help we cannot be here today!</a></h5>-->
-
that could be used to distinguish between the total amount of DNA on the feces of normal and
+
-
diseased patients.</p>
+
-
<br>
+
                        <p>As a proxy for the detection of L-DNA, we decided to build first a generic DNA sensor
 +
                        that could be used to distinguish between the total amount of DNA on the feces of normal and
 +
                        diseased patients.</p>
-
<p>We wanted the reporter to be easily measurable on a lab, but also something that
+
                        <br>
-
could be detected by a patient without any equipment. Thus, our choice was to use a split
+
-
version of mChery Red Fluorescent Protein; mCherry’s red fluorescence can be quantified with
+
-
ease on a fluorometer, and it can also be seen by the naked eye, since its emission fall on the
+
-
visible spectrum. Its split version (which we call hemiCherrys A and B) work on a bimolecular
+
-
complementation system [1] – that is, each of the two parts must be attached to other
+
-
domains that can bring themselves to be close to each other, in a way that the function of the
+
-
reporter is restored and allows for the fluorescence to be emitted after excitation. The
+
-
separated parts do not emit fluorescence nor are able to restore complementarity by
+
-
themselves.</p><br>
+
-
<p>To bring hemiCherrysA and B close to each other on a DNA molecule, we needed them
+
                        <p>We wanted the reporter to be easily measurable on a lab, but also something that
-
to be attached to DNA-Binding domains that would bind close to each other on the substrate
+
                        could be detected by a patient without any equipment. Thus, our choice was to use a split
-
molecule, and also on a spread-manner fashion throughout the spectrum of DNA fragments.  
+
                        version of mChery Red Fluorescent Protein; mCherry’s red fluorescence can be quantified with
-
To accomplish that, we designed a TALE Domain using modified parts from the Freiburg 2012
+
                        ease on a fluorometer, and it can also be seen by the naked eye, since its emission fall on the  
-
collection [2] to bind to the GT (or CA) microsatellite – the most frequent non-single base
+
                        visible spectrum. Its split version (which we call hemiCherrys A and B) work on a bimolecular
-
repetitive DNA on the human genome [3]. Our designed TALE domain binds to a 12-base
+
                        complementation system [1] – that is, each of the two parts must be attached to other
-
region containing six repetitions of the dinucleotide GT. Thus, on a longer DNA fragment
+
                        domains that can bring themselves to be close to each other, in a way that the function of the
-
spanning the microsatellite, two TALE domains can bind close to each other, each attached to  
+
                        reporter is restored and allows for the fluorescence to be emitted after excitation. The
-
a part of hemiCherry, restoring their functionality.</p><br>
+
                        separated parts do not emit fluorescence nor are able to restore complementarity by
 +
                        themselves.</p><br>
-
<p>Of course, before this chimeric proteins can bind to the DNA on the feces, they must
+
                        <p>To bring hemiCherrysA and B close to each other on a DNA molecule, we needed them
-
be secreted by the yeast or bacteria into the extracellular media. For that, we added a  
+
                        to be attached to DNA-Binding domains that would bind close to each other on the substrate
-
secretion signaling domain, according to the chassis: for gram-negative bacteria, we added the  
+
                        molecule, and also on a spread-manner fashion throughout the spectrum of DNA fragments.  
-
TorA twin-arginine translocase domain, a Type II-secretion system [4], wheras for yeast we
+
                        To accomplish that, we designed a TALE Domain using modified parts from the Freiburg 2012
-
used an exportation signaling domain from the Phosphatase-1 protein [5]. Two eleven-amino
+
                        collection [2] to bind to the GT (or CA) microsatellite – the most frequent non-single base
-
acid linker are used for avoiding allosteric interference between the three main domains.
+
                        repetitive DNA on the human genome [3]. Our designed TALE domain binds to a 12-base
-
For a higher expression of the chimeric sensor, the coding region was inserted after a
+
                        region containing six repetitions of the dinucleotide GT. Thus, on a longer DNA fragment
-
strong constitutive promoter, in conjunction with appropriate RBS and terminators.</p><br>
+
                        spanning the microsatellite, two TALE domains can bind close to each other, each attached to
 +
                        a part of hemiCherry, restoring their functionality.</p><br>
-
<div style = "margin: 0 auto; align: center;"><img src="https://static.igem.org/mediawiki/2014/b/bb/UFMG_Brazil2014_parts_design.png" width=900px height=358px></img></div>
+
                        <p>Of course, before this chimeric proteins can bind to the DNA on the feces, they must
 +
                        be secreted by the yeast or bacteria into the extracellular media. For that, we added a
 +
                        secretion signaling domain, according to the chassis: for gram-negative bacteria, we added the
 +
                        TorA twin-arginine translocase domain, a Type II-secretion system [4], wheras for yeast we
 +
                        used an exportation signaling domain from the Phosphatase-1 protein [5]. Two eleven-amino
 +
                        acid linker are used for avoiding allosteric interference between the three main domains.
 +
                        For a higher expression of the chimeric sensor, the coding region was inserted after a
 +
                        strong constitutive promoter, in conjunction with appropriate RBS and terminators.</p><br>
 +
                      <br>
-
<p>References</p>
+
                      <div style="text-align:center;">
-
<br>
+
                        <img class="reflect" style="width:80%;" src="https://static.igem.org/mediawiki/2014/6/6e/Polaroid-part.png"/>
 +
                      </div>
 +
                      <br>
-
<p>[1]: Fan JY, Cui ZQ, Wei HP, Zhang ZP, Zhou YF, Wang YP, Zhang XE. Split mCherry as a new red
+
<table class="tg">
-
bimolecular fluorescence complementation system for visualizing protein-protein interactions
+
  <tr>
-
in living cells. Biochem Biophys Res Commun. 2008 Feb 29;367(1):47-53. Epub 2007 Dec 26.  
+
    <th class="tg-eyl8">Name</th>
-
PubMed PMID: 18158915.</p>
+
    <th class="tg-eyl8">Type</th>
 +
    <th class="tg-eyl8">Description</th>
 +
    <th class="tg-eyl8">Designer</th>
 +
    <th class="tg-eyl8">Length</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514000">BBa_K1514000</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">hemiCherry_A</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">483</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514001">BBa_K1514001</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">hemiCherry_B</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">237</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514002">BBa_K1514002</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">TAL-Protein_GT1_Direpeat_RFC25</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">214</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514003">BBa_K1514003</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">TAL-Protein_GT6_Direpeat_RFC25</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">212</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514004">BBa_K1514004</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">PHO1 Yeast Secretion Domain</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">66</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1514005">BBa_K1514005</a></td>
 +
    <td class="tg-031e">Protein_Domain</td>
 +
    <td class="tg-031e">TOR-A TAT signaling domain for bacteria</td>
 +
    <td class="tg-031e">Carlos Goncalves</td>
 +
    <td class="tg-031e">159</td>
 +
  </tr>
 +
</table>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
-
<p>[2]: https://2012.igem.org/Team:Freiburg</p>
+
      <div class="bg2 p29">
 +
            <div class="container">
 +
                <div class="row">
 +
                    <div class="grid_12 wrap">
 +
                        <div class="thumbnail">
 +
                          <h3>References</h3>
 +
                            <div style="text-align: left; font-size: 80%;">
 +
                              <br>
 +
                            <p><span>[1]: Fan JY, Cui ZQ, Wei HP, Zhang ZP, Zhou YF, Wang YP, Zhang XE. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells. Biochem Biophys Res Commun. 2008 Feb 29;367(1):47-53. Epub 2007 Dec 26. PubMed PMID: 18158915.</span></p>
 +
                            <br><br>
 +
                            <p><span>[2]: https://2012.igem.org/Team:Freiburg</span></p>
 +
                            <br><br>
 +
                            <p><span>[3]: Nadir E, Margalit H, Gallily T, Ben-Sasson SA. Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6470-5. PubMed PMID: 8692839; PubMed Central PMCID: PMC39047.</span></p>
 +
                            <br><br>
 +
                            <p><span>[4]: Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol. 2001 Jan;39(1):47-53. PubMed PMID: 11123687.</span></p>
 +
                            <br><br>
 +
                            <p><span>[5] Hashimoto Y, Koyabu N, Imoto T. Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors. Protein Eng. 1998 Feb;11(2):75-7. PubMed PMID: 9605540.</span></p>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
</div>
-
<br>
+
  <!--========================================================
 +
                            FOOTER
 +
  =========================================================-->
-
<p>[3]: Nadir E, Margalit H, Gallily T, Ben-Sasson SA. Microsatellite spreading in the human
+
<footer id="footer">
-
genome: evolutionary mechanisms and structural implications. Proc Natl Acad Sci U S A. 1996
+
      <div class="container">
-
Jun 25;93(13):6470-5. PubMed PMID: 8692839; PubMed Central PMCID: PMC39047.</p>
+
          <div class="row">
 +
              <div class="grid_12">
 +
                  <a class="logo1" href="https://2014.igem.org/Team:UFMG_Brazil">
 +
                    <img src="https://static.igem.org/mediawiki/2014/8/84/Yeastlogo_small_gray.png" alt="">
 +
                  </a>
 +
                  <div class="copy">
 +
                      <span>&nbsp;&copy;</span>
 +
                      <span id="copyright-year"></span>
 +
                      <span>|</span>
 +
                      <span><a href="https://2014.igem.org/Team:UFMG_Brazil">Team UFMG Brazil</a></span>
 +
                  </div>
 +
              </div>     
 +
          </div>
 +
      </div> 
 +
  </footer>
-
<br>
+
    <script src="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/jquery.js&action=raw&ctype=text/javascript"></script>
 +
    <script src="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/jquery-migrate-1.2.1.js&action=raw&ctype=text/javascript
 +
"></script>
 +
    <script src="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/owl.carousel.js&action=raw&ctype=text/javascript
 +
"></script>     
 +
    <script src="https://2014.igem.org/wiki/index.php?title=Template:UFMG_Brazil/script.js&action=raw&ctype=text/javascript
 +
"></script>
-
<p>[4]: Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein
+
    <meta name="viewport" content="width=device-width,initial-scale=1.0,user-scalable=0">
-
to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol
+
    <script>
-
Microbiol. 2001 Jan;39(1):47-53. PubMed PMID: 11123687.</p>
+
        $(document).ready(function() {
 +
            $("#owl").owlCarousel({
 +
                items: 4,
 +
                itemsDesktop: [1299, 4],
 +
                itemsTablet: [995, 3],
 +
                itemsTabletSmall: [767, 1],
 +
                itemsMobile: [479, 1],
 +
                lazyLoad: true,
 +
                pagination: false,
 +
                navigation: true
 +
            });
 +
        });
 +
        $( document ).ready(function() {
 +
          $('#topmenutoggle').click(function(){
 +
            toggleMenu();
 +
          });
 +
        });
 +
        function toggleMenu(){
 +
          if($('#top-section').css("height") == "25px"){
 +
              $('#top-section').css("height", "10px");
 +
              $('.left-menu').hide();
 +
              $('.right-menu').hide();
 +
          }
 +
          else{
 +
              $('#top-section').css("height", "25px");
 +
              $('.left-menu').show();
 +
              $('.right-menu').show();
 +
          }
 +
        }
 +
    </script>
-
<br>
+
    <a style="margin-right: -49px; right: 50%; display: none;" href="#" id="toTop" class="fa fa-chevron-up"></a>
-
 
+
</body>
-
<p>[5] Hashimoto Y, Koyabu N, Imoto T. Effects of signal sequences on the secretion of hen
+
-
lysozyme by yeast: construction of four secretion cassette vectors. Protein Eng. 1998
+
-
Feb;11(2):75-7. PubMed PMID: 9605540.</p>
+
-
<br>
+
-
 
+
-
</div>
+
-
</div>
+
</html>
</html>
-
{{UFMG_Brazil/footer}}
 

Latest revision as of 03:07, 18 October 2014

Home UFMG Team

Our building blocks!

As a proxy for the detection of L-DNA, we decided to build first a generic DNA sensor that could be used to distinguish between the total amount of DNA on the feces of normal and diseased patients.


We wanted the reporter to be easily measurable on a lab, but also something that could be detected by a patient without any equipment. Thus, our choice was to use a split version of mChery Red Fluorescent Protein; mCherry’s red fluorescence can be quantified with ease on a fluorometer, and it can also be seen by the naked eye, since its emission fall on the visible spectrum. Its split version (which we call hemiCherrys A and B) work on a bimolecular complementation system [1] – that is, each of the two parts must be attached to other domains that can bring themselves to be close to each other, in a way that the function of the reporter is restored and allows for the fluorescence to be emitted after excitation. The separated parts do not emit fluorescence nor are able to restore complementarity by themselves.


To bring hemiCherrysA and B close to each other on a DNA molecule, we needed them to be attached to DNA-Binding domains that would bind close to each other on the substrate molecule, and also on a spread-manner fashion throughout the spectrum of DNA fragments. To accomplish that, we designed a TALE Domain using modified parts from the Freiburg 2012 collection [2] to bind to the GT (or CA) microsatellite – the most frequent non-single base repetitive DNA on the human genome [3]. Our designed TALE domain binds to a 12-base region containing six repetitions of the dinucleotide GT. Thus, on a longer DNA fragment spanning the microsatellite, two TALE domains can bind close to each other, each attached to a part of hemiCherry, restoring their functionality.


Of course, before this chimeric proteins can bind to the DNA on the feces, they must be secreted by the yeast or bacteria into the extracellular media. For that, we added a secretion signaling domain, according to the chassis: for gram-negative bacteria, we added the TorA twin-arginine translocase domain, a Type II-secretion system [4], wheras for yeast we used an exportation signaling domain from the Phosphatase-1 protein [5]. Two eleven-amino acid linker are used for avoiding allosteric interference between the three main domains. For a higher expression of the chimeric sensor, the coding region was inserted after a strong constitutive promoter, in conjunction with appropriate RBS and terminators.




Name Type Description Designer Length
BBa_K1514000 Protein_Domain hemiCherry_A Carlos Goncalves 483
BBa_K1514001 Protein_Domain hemiCherry_B Carlos Goncalves 237
BBa_K1514002 Protein_Domain TAL-Protein_GT1_Direpeat_RFC25 Carlos Goncalves 214
BBa_K1514003 Protein_Domain TAL-Protein_GT6_Direpeat_RFC25 Carlos Goncalves 212
BBa_K1514004 Protein_Domain PHO1 Yeast Secretion Domain Carlos Goncalves 66
BBa_K1514005 Protein_Domain TOR-A TAT signaling domain for bacteria Carlos Goncalves 159

References


[1]: Fan JY, Cui ZQ, Wei HP, Zhang ZP, Zhou YF, Wang YP, Zhang XE. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells. Biochem Biophys Res Commun. 2008 Feb 29;367(1):47-53. Epub 2007 Dec 26. PubMed PMID: 18158915.



[2]: https://2012.igem.org/Team:Freiburg



[3]: Nadir E, Margalit H, Gallily T, Ben-Sasson SA. Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6470-5. PubMed PMID: 8692839; PubMed Central PMCID: PMC39047.



[4]: Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol. 2001 Jan;39(1):47-53. PubMed PMID: 11123687.



[5] Hashimoto Y, Koyabu N, Imoto T. Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors. Protein Eng. 1998 Feb;11(2):75-7. PubMed PMID: 9605540.