Latest revision as of 00:20, 2 November 2017

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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.

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      <label for="vmcb-d"><a>Experiment</a></label>
       <ul>
-         <li style="padding-bottom: 10px; padding-top: 10px">
+         <li>
          <input type="checkbox" id="vmcb-d1" />
-          <label for="vmcb-d1"><a style="text-align: center;">Bacteria <br>to Human Cells ▼</a></label>
+        <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 Impovement <br>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/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/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: 10px">
+       <li style="padding-bottom: 3px">
-       <input type="checkbox" id="vmcb-d2" />
+       <input type="checkbox" id="vmcb-d3" />
-     <label for="vmcb-d2"><a style="text-align: center;">Human Cells to Bacteria ▼</a></label>
+     <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>
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-         <input type="checkbox" id="vmcb-d3" />
+         <input type="checkbox" id="vmcb-d4" />
-     <label for="vmcb-d3"><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>
+     <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>
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      <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">Integrated <br> Human Practice</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>
@@ Line 131: / Line 145: @@
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">　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>
+     <figcaption style="font-size: 16px">
- and <span style="font-style: italic">log1</span> genes to synthesize iP.
+　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>
-　AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of AHLs, 3OC8HSL (C8) was employed in the assay.
+　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.
-</br>
-　In order to achieve C8-dependent transcription in human cells, a chimeric transcription factor named RelA/NLS/TraR was constructed. This protein is comprised of the transcription activating domain of RelA (a kind of human NF-kB family), nuclear localization signal (NLS), and full-length TraR.This protein can activate transcription from appropriate promoters only in the presence of C8 in human cells (see below for details).
-</br>
-　Isopentenyladenine (iP) is kind of a cytokinin, and we use it as a signal molecule from human to <span style="font-style: italic">E. coli</span> cells and for the cross-kingdom communication. Cytokinins are the signaling molecules (or Phytohormones) that plants produce and play important roles in cell growth and differentiation. In the case of Arabidopsis thaliana, extracellular iP is received by a transmembrane receptor, AHK4. AHK4 has a histidine kinase activity, and binding of iP to AHK4 triggers auto-phosphorylation of AHK4 and the following histidine-to-aspartate phosphorelay. As a consequence, transcription from target genes is induced and/or repressed so that physiological states of plants are changed. The histidine kinase activity of AHK4 has shown to be activated depending on iP even in E. coli cells (Suzuki et al. 2001, Lukáš Spíchal et al. 2004). This fact encouraged us to use iP as a signaling molecule in our project (Read <a href = https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay>AHK4 Assay</a> page).
 </p>
@@ Line 149: / Line 160: @@
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;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>
+     <figcaption style="font-size: 16px">
- 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 A. thaliana, and their products jointly work as iP synthetase.
+　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.
@@ Line 158: / Line 170: @@
        <figure>
        <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">Fig1. Construction of iP synthetase genes</figcaption>
+       <figcaption style="font-size: 16px">Fig. 1 Construction of iP synthetase genes</figcaption>
        </figure>
        </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>
@@ Line 169: / Line 185: @@
      <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">
-     <p style="font-family: Poppins;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.
+     <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>
@@ Line 176: / Line 193: @@
      	<figure>
      	<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">Fig2. Result of the qualitative experiment</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 2 Result of the qualitative experiment</figcaption>
      	</figure>
      	</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>
@@ Line 186: / Line 206: @@
      <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">
-     <p style="font-family: Poppins;font-size: 16px">　We confirmed that the transcription of a<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.
+     <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>
@@ Line 209: / Line 230: @@
      <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">
-     <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
 : 159-165.</p>
 <div style="margin-top:16px">
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-<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 to open and close sidebar
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 }
+</script>
+<script>
 // Modal Image Gallery
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+  $('#wrap').css('display','none');
+  $('#loader-bg ,#loader').height(h).css('display','block');
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+$(window).load(function () { //全ての読み込みが完了したら実行
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+  $('#loader').delay(600).fadeOut(300);
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