Team:UANL Mty-Mexico/Wet lab/Light experiments

From 2011.igem.org

(Difference between revisions)
Line 1: Line 1:
-
{{:Team:UANL_Mty-Mexico/Templates/Banner-LightMachine}}
+
{{:Team:UANL_Mty-Mexico/Templates/Banner-LightTable}}
{{:Team:UANL_Mty-Mexico/Templates/Menu-template}}
{{:Team:UANL_Mty-Mexico/Templates/Menu-template}}
<html>
<html>
Line 21: Line 21:
/-->
/-->
 +
 
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" />
   <title>Team: UANL_Mty-Mexico</title>
   <title>Team: UANL_Mty-Mexico</title>
Line 30: Line 31:
<META NAME="robots" CONTENT="FOLLOW,INDEX">
<META NAME="robots" CONTENT="FOLLOW,INDEX">
    
    
-
<link href="http://www.genobiotec2011.org/iGEMwiki/mainStyle.css" rel="stylesheet" type="text/css">
+
  <link href="http://www.genobiotec2011.org/iGEMwiki/mainStyle.css" rel="stylesheet" type="text/css">
 +
<script type="text/javascript" src="http://www.genobiotec2011.org/iGEMwiki/Scripts/jquery-1.3.2.min.js"></script>
<script type="text/javascript" src="http://www.genobiotec2011.org/iGEMwiki/Scripts/jquery-1.3.2.min.js"></script>
Line 44: Line 46:
<script type="text/javascript" src="http://www.genobiotec2011.org/iGEMwiki/Scripts/slimbox2/js/slimbox2.js"></script>
<script type="text/javascript" src="http://www.genobiotec2011.org/iGEMwiki/Scripts/slimbox2/js/slimbox2.js"></script>
-
 
-
 
-
 
-
 
-
<script>
 
-
    var opts = {slideUpSpeed:500, slideDownSpeed: 200};
 
-
    $(document).ready(function(){
 
-
$('.top-menu').removeClass().addClass('top-menu').addClass('style5');
 
-
      $('ul.s-menu li').click(function(){
 
-
        $(this).parent().find('li').removeClass('selected');
 
-
        $(this).addClass('selected');
 
-
      });
 
-
      $('ul.s-style li').click(function(){
 
-
      $('.top-menu').removeClass().addClass('top-menu').addClass('style'+$(this).attr('rel'));
 
-
      $('.top-menu').removeClass().addClass('top-menu').addClass('style5');
 
-
 
-
        $(".top-menu").slideupmenu(opts); 
 
-
      });
 
-
     
 
-
      $('ul.s-way li[rel="1"]').live('click', function() {
 
-
        opts = {slideUpSpeed:500, slideDownSpeed: 200};
 
-
        $(".top-menu").slideupmenu(opts); 
 
-
      });
 
-
      $('ul.s-way li[rel="2"]').live('click', function() {
 
-
        opts = {slideUpSpeed: 500, slideDownSpeed: 500, ease: "easeOutBounce", stopQueue: true};
 
-
        $(".top-menu").slideupmenu(opts); 
 
-
      });
 
-
      $('ul.s-way li[rel="3"]').live('click', function() {
 
-
        opts = {slideUpSpeed: 1000, slideDownSpeed: 100, ease: "easeOutBack", stopQueue: true};
 
-
        $(".top-menu").slideupmenu(opts); 
 
-
      });
 
-
      $('ul.s-way li[rel="4"]').live('click', function() {
 
-
        opts = {slideUpSpeed: 500, slideDownSpeed: 500, ease:false, stopQueue: false};
 
-
        $(".top-menu").slideupmenu(opts); 
 
-
      });
 
-
      $(".top-menu").slideupmenu(opts); 
 
-
    });
 
-
  </script>
 
-
 
 
Line 92: Line 55:
);
);
   </script>  
   </script>  
-
 
Line 101: Line 63:
<body>
<body>
 +
Line 108: Line 71:
     <div class="br2"></div><div class="br2"></div><div class="br2"></div>             
     <div class="br2"></div><div class="br2"></div><div class="br2"></div>             
     <div id="leftColumn">
     <div id="leftColumn">
 +
     <div id="ColorHeader">
     <div id="ColorHeader">
 +
             WetLab: Light Experiments
             WetLab: Light Experiments
-
    </div>
+
    </div>
<div class="br"></div><div class="br"></div>
<div class="br"></div><div class="br"></div>
<div class="br"></div><div class="br"></div>
<div class="br"></div><div class="br"></div>
-
 
+
      <div id="header-project-column">
-
    <div id="header-project-column">
+
<div class="br2"></div>
-
<div class="br2"></div><div class="br2"></div><div class="br2"></div>             
+
<div class="br2"></div>
-
           Light Experiments
+
<div class="br2"></div>             
-
<div class="br2"></div><div class="br2"></div><div class="br2"></div>             
+
           <a name="Overview"></a>Overview
 +
          <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>             
       </div>
       </div>
-
      
+
<p>
 +
Light experiments were planed to demonstrate the function of HuBac community,  controlling expression of five different products through a simple light-based code.
 +
 
 +
This experiments mainly consisted as follows:
 +
<ol>
 +
<li>Testing the function of our light inducing device (<a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Contributions/Light_Machine" title="Light Machine">Light Machine</a>) by using original Dr. Jeff Tabor's plasmids.</li>
 +
<li>2.- Trials of the HuBac community function using "the simplest version of the circuit" which was designed to demonstrate that biphasic switch works as expected.</li>
 +
</ol>
 +
Also, another experiments were planed to test our new <a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Contributions/Parts">parts</a>. Each of them are detailed in the next paragraphs.
 +
</p>
 +
 
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
<div id="header-project-column">
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>         
 +
        <a name="LightMachine"></a>Light Machine Tests
 +
        <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
</div>
 +
<div class="br"></div>
 +
<div class="br"></div>
 +
<span class="subtitle">
 +
Preparing light-inducible <i>E. coli</i> JT2 cells.
 +
</span>
 +
 
 +
<p>
 +
For testing that Light Machine worked as expected,  light-inducible JT2 cells were transformed with different plasmids as specified in the next table:
 +
</p>
 +
<center>
 +
<table width="680px">
 +
  <tr class="yellow">
 +
     <td class="width" align="center">Cell</td>
 +
    <td class="adjacent">Description</td>
 +
    <td class="adjacent">Plasmids</td>
 +
    <td class="adjacent">Constructed</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="center">Green Cell</td>
 +
    <td class="adjacent">Responds to green-light producing LacZ</td>
 +
    <td class="adjacent">pJT122, pPLPCB(S)</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="center">Green Cell (-)</td>
 +
    <td class="adjacent">Does not respond to green light due to absence of chromophore. Used as negative control for Green Cell.</td>
 +
    <td class="adjacent">pJT122</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="center">Red Cell</td>
 +
    <td class="adjacent">Responds to red-light producing LacZ</td>
 +
    <td class="adjacent">pCph8, pJT106, pPLPCB(S)</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
  <td class="bold" align="center">Red Cell (-)</td>
 +
    <td class="adjacent">Does not respond to green light due to absence of chromophore. Used as negative control for Red Cell.</td>
 +
    <td class="adjacent">pCph8, pJT106</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="center">RedGreen Cell</td>
 +
    <td class="adjacent">Responds to red OR green light producing LacZ.</td>
 +
    <td class="adjacent">pJT122, pCph8, pPLPCB(S)</td>
 +
    <td class="adjacent">------</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="center">RedGreen Cell (-)</td>
 +
    <td class="adjacent">Does not respond to green light due to absence of chromophore. Used as negative control for RedGreen Cell.</td>
 +
    <td class="adjacent">pJT122, pCph8</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
</table>
 +
</center> 
 +
<div class="br"></div>
 +
<div class="br"></div>
 +
<div class="br"></div>
 +
<div class="br"></div>
 +
 
 +
 
 +
 
 +
<center>
 +
<div class = "img-holder" style="width:620px">
 +
<a href="https://static.igem.org/mediawiki/2011/1/1d/Bitacora-toWiki.056.png" rel="lightbox" title="Characterization of multiple plasmids transformed into <i>E. coli</i> JT2 strain">
 +
<img src="https://static.igem.org/mediawiki/2011/1/1d/Bitacora-toWiki.056.png" width="596" height="443" alt="pTKRED into JT2" align="center">
 +
</a>
 +
<span class="img-holder-text"><b>Characterization of multiple plasmids transformed into <i>E. coli</i> JT2 strain</b></span>
 +
</div>
 +
</center>
 +
 
 +
 
 +
<span class="subtitle">
 +
Testing Light Machine
 +
</span>
 +
<p>
 +
Green Cells were plated on LB agar media and incubated overnight in absence of light. After that, plates were exposed to green light (532 nm) during 6 hours. Individual colonies were picked up, resuspended on 5 uL of H20 by vortex. After this,10 uL of X-gal were added (20 mg/mL), and there was an incubation period of 30 minutes at 37ºC, finally samples were centrifuged for precipitating cells.
 +
</p>
 +
<p>
 +
Negative control used in this case was a duplicate culture of Green Cells that was all-time covered for avoiding light induction.
 +
</p>
 +
<p>
 +
It may be observed a qualitative difference between sample and negative control.
 +
</p>
 +
 
 +
<center>
 +
<div class = "img-holder" style="width:448px">
 +
<a href="https://static.igem.org/mediawiki/2011/d/d4/Bitacora-toWiki.023.png" rel="lightbox" title="Characterization of multiple plasmids transformed into <i>E. coli</i> JT2 strain">
 +
<img src="https://static.igem.org/mediawiki/2011/d/d4/Bitacora-toWiki.023.png" width="448" height="236" alt="pTKRED into JT2" align="center">
 +
</a>
<div class="br"></div>
<div class="br"></div>
-
<div class="br"></div>
+
<span class="img-holder-text"><b>First test of Light Machine.</b> <i>A)</i> Sample exposed to green light, <i>B)</i> Negative control</span>
-
<div class="br"></div>          
+
</div>
-
 
+
</center>
-
    </div>
+
-
   
+
-
    <div id="rightColumn">
+
-
<!--    <div id="social-div">
+
-
 
+
-
<a href="http://twitter.com/iGEMUANL" class="twitter-follow-button" data-show-count="false" data-align="right">Follow @iGEMUANL</a>
+
-
  <script src="http://platform.twitter.com/widgets.js" type="text/javascript"></script>
+
-
        <div class="br"></div>        
+
-
                <iframe src="http://www.facebook.com/plugins/like.php?app_id=134010103347967&amp;href=http%3A%2F%2Fwww.facebook.com%2Fpages%2FiGEM-UANL%2F257596767587618&amp;send=false&amp;layout=button_count&amp;width=200&amp;show_faces=false&amp;action=like&amp;colorscheme=light&amp;font&amp;height=21" scrolling="no" frameborder="0" style="border:none; overflow:hidden; width:80px; height:21px;" allowTransparency="true">
+
-
               
+
-
           
+
 
-
            </iframe>  
+
-
</div>-->
+
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
<div id="header-project-column">
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>         
 +
        <a name="Summary"></a>Summary
 +
        <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div> 
 +
</div>
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
 
 +
 
 +
<p>
 +
<center>
 +
<table width="450px">
 +
  <tr>
 +
    <th colspan="3">Integration procedure</th>
 +
  </tr>
 +
  <tr class="yellow">
 +
    <td align="center">Steps</td>
 +
    <td class="adjacent">Status</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="left">Transforming with helper plasmid pTKRED</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td class="bold" align="left">Integration of Landing Pad</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
    <tr>
 +
    <td class="bold" align="left">Transforming with pTKIP plasmid.</td>
 +
    <td class="adjacent">Done</td>
 +
  </tr>
 +
 
 +
    <tr>
 +
    <td class="bold" align="left">Recombination of pTKIP plasmid with Landind Pad.</td>
 +
    <td class="adjacent">In progress</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
</p>
 +
 
 +
 
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>
 +
<div id="header-project-column">
 +
  <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div>         
 +
        <a name="References"></a>References
 +
        <div class="br2"></div>
 +
<div class="br2"></div>
 +
<div class="br2"></div> 
 +
</div>
 +
 
 +
<ol>
 +
<li>Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs <i>Nucleic Acids Res</i> <b>38</b>:e92.</li>
 +
</ol>
 +
 
 +
 
 +
</div>
      
      
-
       
+
     <div id="rightColumn"> <a href="#top" title="Go back to top"><img src="http://www.genobiotec2011.org/iGEMwiki/images/OurSymbol.png" width="200" height="160" alt="OurSymbol"></a>
-
       
+
-
     <a href="#top"><img src="https://static.igem.org/mediawiki/igem.org/0/09/OurSymbol.png" width="200" height="160" alt="OurSymbol"></a>
+
      
      
  <div class="br"></div>
  <div class="br"></div>
         <div class="br"></div>
         <div class="br"></div>
 +
         <div class="br"></div>
         <div class="br"></div>
     <div id="botonera">
     <div id="botonera">
-
     <div class="lateral-button"><a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Project/The_Code">The Code</a></div>
+
 
-
     <div class="lateral-button"><a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Project/Mechanism">Mechanism</a></div>
+
     
-
    <div class="lateral-button"><a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Project/Circuit">Genetic Circuit</a></div>
+
     <div class="lateral-button"><a href="#Overview">Overview</a></div>
-
    <div class="lateral-button"><a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Project/Applications">Applications</a></div>
+
     <div class="lateral-button"><a href="#Procedure">Procedure</a></div>
-
 
+
    <div class="lateral-button"><a href="#Summary">Summary</a></div>
 +
    <div class="lateral-button"><a href="#References">References</a></div>
     </div>
     </div>
Line 169: Line 321:
   </div>
   </div>
 +
      
      
     <div style="clear:both"></div> <!-- Catches the shorter column up -->
     <div style="clear:both"></div> <!-- Catches the shorter column up -->
      
      
   </div>
   </div>
-
   <p><img src="https://static.igem.org/mediawiki/igem.org/4/49/SymbolFooter.png" width="950" height="70" alt="OurSymbol"></p>
+
   <p><img src="http://www.genobiotec2011.org/iGEMwiki/images/SymbolFooter.png" width="950" height="70" alt="OurSymbol"></p>
</div>
</div>
</div>
</div>
 +
 +
     
</body>
</body>
</html>
</html>
{{:Team:UANL_Mty-Mexico/Templates/Footer-template}}
{{:Team:UANL_Mty-Mexico/Templates/Footer-template}}

Revision as of 00:29, 29 September 2011

banner-main iGEM-logo
Team: UANL_Mty-Mexico
Team: UANL_Mty-Mexico
WetLab: Light Experiments
Overview

Light experiments were planed to demonstrate the function of HuBac community, controlling expression of five different products through a simple light-based code. This experiments mainly consisted as follows:

  1. Testing the function of our light inducing device (Light Machine) by using original Dr. Jeff Tabor's plasmids.
  2. 2.- Trials of the HuBac community function using "the simplest version of the circuit" which was designed to demonstrate that biphasic switch works as expected.
Also, another experiments were planed to test our new parts. Each of them are detailed in the next paragraphs.

Light Machine Tests
Preparing light-inducible E. coli JT2 cells.

For testing that Light Machine worked as expected, light-inducible JT2 cells were transformed with different plasmids as specified in the next table:

Cell Description Plasmids Constructed
Green Cell Responds to green-light producing LacZ pJT122, pPLPCB(S) Done
Green Cell (-) Does not respond to green light due to absence of chromophore. Used as negative control for Green Cell. pJT122 Done
Red Cell Responds to red-light producing LacZ pCph8, pJT106, pPLPCB(S) Done
Red Cell (-) Does not respond to green light due to absence of chromophore. Used as negative control for Red Cell. pCph8, pJT106 Done
RedGreen Cell Responds to red OR green light producing LacZ. pJT122, pCph8, pPLPCB(S) ------
RedGreen Cell (-) Does not respond to green light due to absence of chromophore. Used as negative control for RedGreen Cell. pJT122, pCph8 Done
pTKRED into JT2 Characterization of multiple plasmids transformed into E. coli JT2 strain
Testing Light Machine

Green Cells were plated on LB agar media and incubated overnight in absence of light. After that, plates were exposed to green light (532 nm) during 6 hours. Individual colonies were picked up, resuspended on 5 uL of H20 by vortex. After this,10 uL of X-gal were added (20 mg/mL), and there was an incubation period of 30 minutes at 37ºC, finally samples were centrifuged for precipitating cells.

Negative control used in this case was a duplicate culture of Green Cells that was all-time covered for avoiding light induction.

It may be observed a qualitative difference between sample and negative control.

pTKRED into JT2
First test of Light Machine. A) Sample exposed to green light, B) Negative control
Summary

Integration procedure
Steps Status
Transforming with helper plasmid pTKRED Done
Integration of Landing Pad Done
Transforming with pTKIP plasmid. Done
Recombination of pTKIP plasmid with Landind Pad. In progress

References
  1. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res 38:e92.

OurSymbol

Team: UANL_Mty-Mexico

Retrieved from "http://2011.igem.org/Team:UANL_Mty-Mexico/Wet_lab/Light_experiments"