Difference between revisions of "Team:TokyoTech/Experiment/Chimeric Transcription Factor"

(Created page with "{{TokyoTech/css/reset}} {{TokyoTech/css/main}} <!DOCTYPE html> <html> <head> <title>Coli Sapiens</title> <meta charset="UTF-8"> <meta name="viewport" content="width=device-wid...")
 
 
(39 intermediate revisions by 4 users not shown)
Line 3: Line 3:
 
<!DOCTYPE html>
 
<!DOCTYPE html>
 
<html>
 
<html>
−
<head>
 
 
<title>Coli Sapiens</title>
 
<title>Coli Sapiens</title>
 
<meta charset="UTF-8">
 
<meta charset="UTF-8">
 
<meta name="viewport" content="width=device-width, initial-scale=1">
 
<meta name="viewport" content="width=device-width, initial-scale=1">
 
<link rel="stylesheet" href="main.css">
 
<link rel="stylesheet" href="main.css">
−
<link rel="stylesheet" href="https://fonts.googleapis.com/css?family=Poppins">
 
 
<style>
 
<style>
−
body,h1,h2,h3,h4,h5 {font-family: "Poppins", sans-serif}
+
body,h1,h2,h3,h4,h5 {font-family: "Arial", sans-serif}
 
body {font-size:16px;}
 
body {font-size:16px;}
 
.w3-half img{margin-bottom:-6px;margin-top:16px;opacity:0.8;cursor:pointer}
 
.w3-half img{margin-bottom:-6px;margin-top:16px;opacity:0.8;cursor:pointer}
 
.w3-half img:hover{opacity:1}
 
.w3-half img:hover{opacity:1}
 
</style>
 
</style>
−
<head>
 
 
<body>
 
<body>
 +
 +
<div id="loader-bg">
 +
  <div id="loader">
 +
    <div class="photo-show">
 +
    <img src="https://static.igem.org/mediawiki/2017/f/f4/T--TokyoTech--load_1.png" width="600">
 +
    <img src="https://static.igem.org/mediawiki/2017/4/43/T--TokyoTech--load_2.png" width="600">
 +
    </div>
 +
  </div>
 +
</div>
  
 
<!-- Sidebar/menu -->
 
<!-- Sidebar/menu -->
 
<nav class="w3-sidebar w3-red w3-collapse w3-top w3-large w3-padding" style="z-index:3;width:300px;font-weight:bold;" id="mySidebar"><br>
 
<nav class="w3-sidebar w3-red w3-collapse w3-top w3-large w3-padding" style="z-index:3;width:300px;font-weight:bold;" id="mySidebar"><br>
−
   <a href="javascript:void(0)" onclick="w3_close()" class="w3-button w3-hide-large w3-display-topleft" style="width:100%;font-size:22px">Close Menu</a>
+
   <a href="javascript:void(0)" onclick="w3_close()" class="w3-button w3-hide-large w3-display-topleft" style="width:100%;font-size:20px">Close Menu</a>
−
   <div class="w3-container">
+
   <div class="w3-container" style="margin-top: 20px;margin-bottom: 25px" id="wrap">
−
     <img src="https://static.igem.org/mediawiki/2017/5/57/T--TokyoTech--logo2.png" style="width: 100%">
+
     <img src="https://static.igem.org/mediawiki/2017/a/a8/T--TokyoTech--logo_white_bright_10211603.png" style="width: 100%">
 
   </div>
 
   </div>
−
   <div class="w3-bar-block" style="padding-top: 15px">
+
 
−
     <ul id="normal" class="dropmenu">
+
   <div style="padding-top: 15px; padding-left: 25px; id="wrap">
−
     <li><a href="#overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Home</a>  
+
 
 +
  <div id="contents" style="z-index: 10">
 +
 
 +
    <ul class="click_menu_checkbox vertm_down">
 +
    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px;">
 +
    <input type="checkbox" id="vmcb-a" />
 +
    <label for="vmcb-a"><a href="https://2017.igem.org/Team:TokyoTech">Home</a></label>
 +
    </li>
 +
    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-b" />
 +
    <label for="vmcb-b"><a href="https://2017.igem.org/Team:TokyoTech/Medal">Achievements</a></label>
 +
    </li>
 +
    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-c" />
 +
    <label for="vmcb-c"><a>Project</a></label>
 +
    <ul>
 +
      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Description" class="w3-bar-item w3-button w3-hover-white">Description</a></li>
 +
      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Project/Basic_Parts" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Basic Parts</a></li>
 +
      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Project/Composite_Parts" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Composite Parts</a></li>
 +
     </ul>
 +
    </li>
 +
 
 +
    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-d" />
 +
    <label for="vmcb-d"><a>Experiment</a></label>
 +
    <ul>
 +
 
 +
        <li>
 +
        <input type="checkbox" id="vmcb-d1" />
 +
        <label for="vmcb-d1"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" style="text-align: center;">Overview</a></label>
 +
        </li>
 +
 
 +
        <li style="padding-bottom: 10px; padding-top: 5px">
 +
        <input type="checkbox" id="vmcb-d2" />
 +
          <label for="vmcb-d2"><a style="text-align: center;">Bacteria to <br>Human Cells ▼</a></label>
 +
            <ul>
 +
              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">TraI Assay</a></li>
 +
              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">TraI Improvement <br>Assay</a></li>
 +
              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraR_Reporter_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" >TraR Reporter <br>Assay</a></li>
 +
              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Transcriptome_Analysis" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Transcriptome <br>Analysis</a></li>
 +
              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Chimeric_Transcription_Factor" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Chimeric <br> Transcription <br> Factor Assay</a></li>
 +
            </ul>
 +
      </li>
 +
 
 +
      <li style="padding-bottom: 3px">
 +
      <input type="checkbox" id="vmcb-d3" />
 +
     <label for="vmcb-d3"><a style="text-align: center;">Human Cells to <br>Bacteria ▼</a></label>
 +
        <ul>
 +
          <li><a href="https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">AHK4 Assay</a></li>
 +
          </ul>
 +
        </li>
 +
 
 +
        <li>
 +
        <input type="checkbox" id="vmcb-d4" />
 +
    <label for="vmcb-d4"><a href="https://2017.igem.org/Team:TokyoTech/InterLab" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" style="text-align: center;">InterLab</a></label>
 +
        </li>
 +
    </ul>
 +
    </li>
 +
 
 +
 
 +
    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-e" />
 +
    <label for="vmcb-e"><a href="https://2017.igem.org/Team:TokyoTech/Model">Modelling</a></label>
 +
    </li>
 +
 
 +
    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-f" />
 +
    <label for="vmcb-f"><a>Human Practices</a></label>
 
     <ul>
 
     <ul>
−
     <li><a href="#overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">1</a></li>
+
     <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Overview</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP/Silver" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Silver</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP/Gold_Integrated" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Gold (Integrated)</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Demonstrate" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Demonstrate</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Collaborations" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Collaborations</a></li>
 
     </ul>
 
     </ul>
 
     </li>
 
     </li>
 +
 +
    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
 +
    <input type="checkbox" id="vmcb-g" />
 +
    <label for="vmcb-g"><a>About us</a></label>
 +
    <ul>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Team" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Team</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Attributions" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Attributions</a></li>
 +
    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Sponsors" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Sponsors</a></li>
 
     </ul>
 
     </ul>
−
     <a href="#project" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Project</a>  
+
     </li>
−
     <a href="#modeling" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Modeling</a>  
+
     </ul>
−
     <a href="#hp" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Human Practices</a>  
+
     </div>
−
    <a href="#notebook" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Notebook</a>
+
 
−
    <a href="#team" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Team</a>
+
−
    <a href="#sponsers" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Sponsers</a>
+
 
   </div>
 
   </div>
 
</nav>
 
</nav>
Line 53: Line 136:
  
 
<div class="w3-container" id="contact" style="margin-top:30px"><!-- ページタイトル -->
 
<div class="w3-container" id="contact" style="margin-top:30px"><!-- ページタイトル -->
−
<h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">Human Cell Assay</h1>
+
<h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">Chimeric Transcription Factor Assay</h1>
 
</div>
 
</div>
  
Line 62: Line 145:
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
  
−
     <p style="font-family: Poppins;font-size: 16px">In this assay, we checked whether human cells (<span style="font-style: italic">EA.hy926</span> cell) receive AHL, a signaling molecule derived from <i>E. coli</i> and induce the transcription of <i>atlPT4</i> gene and <i>log1</i> gene which synthesize iP. Also, we checked whether IP (Isopentenyl adenine) molecules are synthesized in <span style="font-style: italic">EA.hy926</span> cells by TLC.
+
     <figcaption style="font-size: 16px">
−
    </p>
+
　In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from <span style="font-style: italic">E. coli</span> and induce the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes to synthesize iP(Read <a href = https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay>"AHK4 Assay"</a> page).
−
    <br>
+
</br>
−
    <p style="font-family: Poppins;font-size: 16px"><u>Note</u></p>
+
　AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”(Read <a href = https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay>"TraI Assay"</a> page). Among several kinds of AHLs, 3OC8HSL (C8) was chosen in the assay.  
−
    <p style="font-family: Poppins;font-size: 16px">AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of the signaling molecules, we used 3OC8AHL(C8) in the assay.</p>
+
 
 +
</p>
  
 
   </div>
 
   </div>
Line 76: Line 160:
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
  
−
     <p style="font-family: Poppins;font-size: 16px">As shown in Fig. , we introduced two kinds of constructs into <span style="font-style: italic">EA.hy926</span> cells by electroporation. CAG promoter constantly expresses relA/NLS/traR. After the protein binds with 3OC8AHL(C8), the complex exposes nuclear localization signal and activates CMVmin promoter in CMVmin_atIPT4_IVS_IRES_LOG1_polyA. As a result of pCMVmin's activation, downstream <i>atlPT4</i> gene and <i>log1</i> gene are transcripted. <i>atlPT4</i> gene and <i>log1</i> gene jointly work as IP synthetase.
+
     <figcaption style="font-size: 16px">
 +
　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)<sub>7</sub> sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)<sub>7</sub> sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)<sub>7</sub> is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the <span style="font-style: italic">atIPT4</span>
 +
 and <span style="font-style: italic">log1</span> genes are transcribed depending on C8. The <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes are derived from <span style="font-style: italic">A. thaliana</span>, and their products jointly work as iP synthetase.
 +
 
 +
 
 
     </p>
 
     </p>
  
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
−
       <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:50%">
+
       <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:75%">
−
       <figcaption style="font-family: Poppins;font-size: 16px">Fig. 画像タイトル</figcaption>
+
       <figcaption style="font-size: 16px">Fig. 1 Construction of iP synthetase genes</figcaption>
 
       </figure>
 
       </figure>
 
       </div>
 
       </div>
 +
    <figcaption style="font-size: 16px">
 +
　IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent manner. The term “polyA” indicates the polyadenylation signal that  is important for the nuclear export, translation, and stability of mRNA.
 +
</p>
 +
  
 
   </div>
 
   </div>
Line 93: Line 185:
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Results</b></h1><!-- 小見出し -->
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Results</b></h1><!-- 小見出し -->
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
−
     <p style="font-family: Poppins;font-size: 16px">文章
+
     <figcaption style="font-size: 16px">
 +
　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.
 +
 
 
     </p>
 
     </p>
  
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
     <figure>
 
     <figure>
−
     <img src="https://static.igem.org/mediawiki/2017/f/f9/T--TokyoTech--human_cell_results_1022.png" style="max-width:50%">
+
     <img src="https://static.igem.org/mediawiki/2017/e/e9/Human_cell_result_v3.png" style="max-width:85%">
−
     <figcaption style="font-family: Poppins;font-size: 16px">Fig. 画像タイトル</figcaption>
+
     <figcaption style="font-size: 16px">Fig. 2 Result of the qualitative experiment</figcaption>
 
     </figure>
 
     </figure>
 
     </div>
 
     </div>
 +
    <figcaption style="font-size: 16px">
 +
　The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.
 +
</p>
 
   </div>
 
   </div>
  
Line 109: Line 206:
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
−
     <p style="font-family: Poppins;font-size: 16px">We checked the transcription of <i>atlPT4</i> gene and <i>log1</i> gene are induced by C8 addition and the induction depends on C8 concentration.
+
     <figcaption style="font-size: 16px">
 +
　We confirmed that the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes were induced by C8 addition and the degree of induction depends on C8 concentration.
 +
 
 
     </p>
 
     </p>
 +
 +
</div>
 +
 +
<hr>
 +
 +
<div class="w3-container" id="results" style="margin-top:20px">
 +
    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Protocol</b></h1>
 +
    <hr style="width:50px;border:5px solid red" class="w3-round">
 +
 +
    <center>
 +
      <object data="https://static.igem.org/mediawiki/2017/c/ca/Chimeric_Transcription_Factor_Assay_Protocol_v2.pdf" type="application/pdf" style="width: 600px; height: 300px"></object>
 +
    </center>
  
 
</div>
 
</div>
Line 119: Line 230:
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Reference</b></h1>
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Reference</b></h1>
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
 
     <hr style="width:50px;border:5px solid red" class="w3-round">
−
     <p style="font-family: Poppins;font-size: 16px"><p>Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL  
+
     <figcaption style="font-size: 16px">Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL  
 
4: 159-165.</p>
 
4: 159-165.</p>
 
<div style="margin-top:16px">
 
<div style="margin-top:16px">
Line 128: Line 239:
 
</div><!-- ここまでが編集可能範囲 -->
 
</div><!-- ここまでが編集可能範囲 -->
  
−
<div class="w3-light-grey w3-container w3-padding-32" style="margin-top:75px;padding-right:58px"><p class="w3-right">Hajime Fujita:  <a href="96haji.me" title="W3.CSS" target="_blank" class="w3-hover-opacity">All Rights Reserved</a></p></div>
+
<div class="w3-container" id="contact" style="margin-top:20px">
 +
<p id="pageTop" style="text-align:right"><a href="#wrap"><img src="https://static.igem.org/mediawiki/2017/0/0d/T--TokyoTech--page_top_2.png" style="width:200px"></a></p>
 +
</div>
 +
 
 +
<!-- W3.CSS Container -->
 +
<div class="w3-light-grey w3-container w3-padding-32" style="margin-top:75px;padding-right:58px"><p class="w3-right"><a href="http://96haji.me/" title="W3.CSS" target="_blank" class="w3-hover-opacity">Hajime Fujita with W3.CSS: All Rights Reserved</a></p></div>
  
 +
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.1/jquery.min.js"></script>
 
<script>
 
<script>
 
// Script to open and close sidebar
 
// Script to open and close sidebar
Line 141: Line 258:
 
     document.getElementById("myOverlay").style.display = "none";
 
     document.getElementById("myOverlay").style.display = "none";
 
}
 
}
−
 
+
</script>
 +
<script>
 
// Modal Image Gallery
 
// Modal Image Gallery
 
function onClick(element) {
 
function onClick(element) {
Line 149: Line 267:
 
   captionText.innerHTML = element.alt;
 
   captionText.innerHTML = element.alt;
 
}
 
}
 +
$(function() {
 +
  var h = $(window).height();
 +
 +
  $('#wrap').css('display','none');
 +
  $('#loader-bg ,#loader').height(h).css('display','block');
 +
});
 +
 +
$(window).load(function () { //全ての読み込みが完了したら実行
 +
  $('#loader-bg').delay(900).fadeOut(800);
 +
  $('#loader').delay(600).fadeOut(300);
 +
  $('#wrap').css('display', 'block');
 +
});
 
</script>
 
</script>
 +
<script>
 +
//■page topボタン
 +
$(function(){
 +
var topBtn=$('#pageTop');
 +
topBtn.hide();
 +
 +
//◇ボタンの表示設定
 +
$(window).scroll(function(){
 +
  if($(this).scrollTop()>80){
 +
    //---- 画面を80pxスクロールしたら、ボタンを表示する
 +
    topBtn.fadeIn();
 +
  }else{
 +
    //---- 画面が80pxより上なら、ボタンを表示しない
 +
    topBtn.fadeOut();
 +
  }
 +
});
 +
 +
// ◇ボタンをクリックしたら、スクロールして上に戻る
 +
topBtn.click(function(){
 +
  $('body,html').animate({
 +
  scrollTop: 0},500);
 +
  return false;
 +
});
 +
 +
});
 +
</script>
 +
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 00:20, 2 November 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

Chimeric Transcription Factor Assay

Introduction

　In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atIPT4 and log1 genes to synthesize iP(Read "AHK4 Assay" page).
　AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”(Read "TraI Assay" page). Among several kinds of AHLs, 3OC8HSL (C8) was chosen in the assay.

Summary

　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)7 sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)7 sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)7 is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the atIPT4  and log1 genes are transcribed depending on C8. The atIPT4 and log1 genes are derived from A. thaliana, and their products jointly work as iP synthetase.

Fig. 1 Construction of iP synthetase genes
　IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent manner. The term “polyA” indicates the polyadenylation signal that is important for the nuclear export, translation, and stability of mRNA.

Results

　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of atIPT4 and log1 was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.

Fig. 2 Result of the qualitative experiment
　The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.

Discussion

　We confirmed that the transcription of atIPT4 and log1 genes were induced by C8 addition and the degree of induction depends on C8 concentration.

Protocol

Reference

Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.

Hajime Fujita with W3.CSS: All Rights Reserved