Team/CINVESTAV-IPN-UNAM MX/Metods.htm

From 2012.igem.org

(Difference between revisions)
 
(14 intermediate revisions not shown)
Line 1: Line 1:
-
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN"
+
<html xmlns="http://www.w3.org/1999/xhtml">
-
"http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
+
<head>
-
<!-- saved from url=(0014)about:internet -->
+
<meta http-equiv="content-type" content="text/html; charset=utf-8" />
-
<html xmlns="http://www.w3.org/1999/xhtml"><head>
+
<title>Rho</title>
-
<title>Home</title>
+
<style type="text/css">
 +
#top-section {
 +
position: relative;
 +
height: 20px;
 +
width: 975px;
 +
margin: 0 auto 0 auto;
 +
border-left: hidden;
 +
border-right: hidden;
 +
border-bottom: hidden;
 +
}
-
<link rel="stylesheet" href="https://dl.dropbox.com/u/199422/Cinvestav/style.css" />
+
* {
 +
        margin: 0;
 +
        padding: 0;
 +
}
-
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
+
body {
-
<style type="text/css">
+
        background: #FFFFFF url(https://static.igem.org/mediawiki/2012/f/f3/Fondo_bod.jpg) repeat-x;
-
d img {display: block;}td img {display: block;}
+
        text-align: justify;
-
</style>
+
        font-family: Georgia, "Times New Roman", Times, serif;
 +
        font-size: 12px;
 +
        color: #000000;
 +
}
-
<!--Fireworks CS6 Dreamweaver CS6 target. Created Tue Sep 25 17:05:18 GMT-0500 (Central Daylight Time (Mexico)) 2012-->
+
h1, h2, h3 {
 +
        color: #333333;
 +
}
 +
 
 +
h1, h2 {
 +
        margin-bottom: 20px;
 +
        font-weight: normal;
 +
}
 +
 
 +
h1 {
 +
        font-size: 197%;
 +
}
 +
 
 +
h2 {
 +
        font-size: 152%;
 +
}
 +
 
 +
h3 {
 +
        font-size: 100%;
 +
}
 +
 
 +
p, ul, ol {
 +
        margin-bottom: 20px;
 +
        line-height: 1.8em;
 +
}
 +
 
 +
ul, ol {
 +
        margin-left: 3em;
 +
}
 +
#text2
 +
{
 +
color: #333333;
 +
font-size: 152%;
 +
margin-bottom: 20px;
 +
font-weight: normal;
 +
}
 +
blockquote {
 +
        padding-left: 3em;
 +
        background: url(https://static.igem.org/mediawiki/2012/1/11/Img08.gif) no-repeat;
 +
        font-style: italic;
 +
}
 +
 
 +
a:link {
 +
        color: #1953A7;
 +
}
 +
 
 +
a:active {
 +
        color: #ED5C0C;
 +
}
 +
 
 +
a:visited {
 +
        color: #666666;
 +
}
 +
 
 +
a:hover {
 +
        text-decoration: none;
 +
        color: #067600;
 +
}
 +
 
 +
img {
 +
        border: none;
 +
}
 +
 
 +
img.left {
 +
        float: left;
 +
        margin: 0 20px 10px 0;
 +
}
 +
 
 +
/* Menu */
 +
 
 +
#menu {
 +
        width: 770px;
 +
        height: 65px;
 +
        margin: 0 auto;
 +
        border-right: 1px solid #EDEBD5;
 +
}
 +
 
 +
#menu ul {
 +
        margin: 0;
 +
        padding: 0;
 +
        list-style: none;
 +
}
 +
 
 +
#menu li {
 +
        display: inline;
 +
        font-size: 12px;
 +
        color:#4E4E4E;
 +
}
 +
 
 +
#menu a {
 +
        display: block;
 +
        float: left;
 +
        width: 95px;
 +
        height: 45px;
 +
        padding: 10px 0 0 0;
 +
        border-left: 1px solid #EDEBD5;
 +
        text-transform: lowercase;
 +
        text-decoration: none;
 +
        text-align: center;
 +
        font-size: 135%;
 +
        color: #BABABA;
 +
}
 +
 
 +
#menu a:hover {
 +
        background: #F7F7F4;
 +
}
 +
 
 +
#menu .active a {
 +
        background: #5dbb10;
 +
        color: #FFFFFF;
 +
}
 +
 
 +
/* Header */
 +
 
 +
#header {
 +
        width: 770px;
 +
        height: 170px;
 +
        margin: 0 auto;
 +
}
 +
 
 +
#header h1 {
 +
        float: left;
 +
        width: 220px;
 +
        height: 100px;
 +
        margin: 0;
 +
        padding: 70px 0 0 0;
 +
        background: url(https://static.igem.org/mediawiki/2012/0/0a/Img02.jpg) no-repeat;
 +
        text-align: center;
 +
        font-size: 36px;
 +
}
 +
 
 +
#header h2 {
 +
        float: right;
 +
        width: 530px;
 +
        height: 86px;
 +
        margin: 0;
 +
        background: url(https://static.igem.org/mediawiki/2012/4/4a/Img03.jpg) no-repeat;
 +
        padding: 84px 20px 0 0;
 +
        text-align: right;
 +
}
 +
 
 +
#header a {
 +
        text-transform: lowercase;
 +
        text-decoration: none;
 +
        font-style: italic;
 +
        font-weight: normal;
 +
        color: #FFFFFF;
 +
}
 +
 
 +
/* Page */
 +
 
 +
#page {
 +
        width: 770px;
 +
        margin: 0 auto;
 +
        padding: 20px 0;
 +
}
 +
#context{
 +
        float: left;
 +
        width: 570px;
 +
        padding-top:5px;
 +
        }       
 +
/* menudecontenido */
 +
 
 +
#sidebar {
 +
        float: right;
 +
        width: 160px;
 +
        padding: 15px;
 +
        background: #EDEBD5;
 +
}
 +
 
 +
#sidebar h2 {
 +
        margin: 0;
 +
        padding: 5px 15px;
 +
        color: #FFFFFF;
 +
}
 +
 
 +
#sidebar h3 {
 +
        font-size: 70%;
 +
        color: #FFFFFF;
 +
}
 +
 
 +
#sidebar ul {
 +
        margin: 0;
 +
        padding: 15px;
 +
        list-style: none;
 +
}
 +
 
 +
#sidebar li {
 +
              font-size: 14px;
 +
}
 +
 
 +
#sidebar a {
 +
        color: #FFFFFF;
 +
}
 +
 
 +
.orangebox {
 +
        background: url(https://static.igem.org/mediawiki/2012/b/b0/Img05.gif) repeat-y;
 +
}
 +
 
 +
.orangebox h2 {
 +
        background: url(https://static.igem.org/mediawiki/2012/c/c2/Img06.gif) repeat-y;
 +
}
 +
 
 +
/* Footer */
 +
 
 +
#piedepagina {
 +
        clear: both;
 +
        height: 150px;
 +
        padding: 25px 0;
 +
        background: #EDEBD5 url(https://static.igem.org/mediawiki/2012/a/a5/Img04.gif) repeat-x;
 +
}
 +
 
 +
#sponsors{
 +
 +
        float: left;
 +
        width: 85px;
 +
        padding-top:5px;
 +
        background: #EDEBD5;
 +
 +
      }
 +
 
 +
.firstHeading {
 +
margin-bottom: .1em;
 +
visibility:hidden;
 +
}
 +
 
 +
img[src*="/wiki/skins/common/images/wiki.png"]{
 +
    width:0;
 +
    height:0px;
 +
    padding-right:975px;
 +
    background:transparent url(https://2011.igem.org/wiki/skins/common/images/wiki.png) top left no-repeat;
 +
}
 +
 
 +
</style>
 +
<meta name="keywords" content="" />
 +
<meta name="description" content="" />
</head>
</head>
-
<body bgcolor="#ffffff">
+
<body>
-
<table style="display: inline-table;" border="0" cellpadding="0" cellspacing="0" width="950" bgcolor="">
+
<div id="menu">
-
<!-- fwtable fwsrc="INDEX.fw.png" fwpage="Page 1" fwbase="principal.jpg" fwstyle="Dreamweaver" fwdocid = "927795023" fwnested="0" -->
+
  <ul>
-
   <tr>
+
    <li><a href="home.htm">Home</a></li>
-
  <td><img src="images/spacer.gif" width="240" height="1" alt="" /></td>
+
    <li><a href="rhodofactory.htm">Rhodofactory</a></li>
-
  <td><img src="images/spacer.gif" width="710" height="1" alt="" /></td>
+
    <li class="active"><a href="promoterselection.htm">Promoter selection </a></li>
-
  <td><img src="images/spacer.gif" width="1" height="1" alt="" /></td>
+
    <li><a href="results.htm">results </a></li>
-
  </tr>
+
    <li><a href="perspectives.htm">Perspectives</a></li>
 +
    <li><a href="Modelling.htm">Modeling</a></li>
 +
    <li><a href="aboutus.htm">About us</a> </li>
 +
    <li><a href="outreach.htm">outreach</a></li>
 +
   </ul>
 +
</div>
 +
<!-- end #menu -->
 +
<div id="header">
 +
  <h1><a href="#"></a></h1>
 +
  <h2><span></span></h2>
 +
</div>
 +
<!-- end #header -->
 +
<div id="page">
 +
  <div id="context">
 +
    <h1><em>Materials and Methods!</em></h1>
 +
    <p id="text2"> Biobicks</p>
 +
    <p>The following biobicks were recuperated from the 2009 and 2012 iGEM distribution plates.
 +
    </p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2012/5/54/Figuram01.jpg" alt="figuram01" width="469" height="259"><br>
 +
    </div><br>
-
  <tr>
+
<p id="text2">Circuits assembly </p>
-
  <td><img src="https://static.igem.org/mediawiki/2012/6/68/LOGOPRIN.jpg" width="240" height="178" alt="Logo Principal" /></td>
+
    <p>Plasmids were obtained by miniprep technique using the kit: Zyppy Plasmid Miniprep Kit and quantified by Nanodrop.</p>
-
  <td bgcolor="01A79D"><ul class="nav">
+
<p>For the constitutive promoters (J23101, J23102, J23104, J23107, J23108, J23111 and J23115) the following enzyme digest were used: 8 hours at 37ºC: </p>
-
<li>
+
<div align="center"><img src="https://static.igem.org/mediawiki/2012/0/01/Figuraam02.jpg" alt="figuram02" width="431" height="169"></div>
-
<a href="home.htm">Home<span class="flecha">&#9660;</span></a>
+
<p><br>
-
</li>
+
After enzyme digestion, a gel was made to confirm the released fragments and the band purification was performed using the kit: Gel DNA Recovery Kit.<br>
-
<li>
+
-
<a href="#">Rhodofactory<span class="flecha">&#9660;</span></a>
+
The biobrick J54103 was extracted from the plate and amplified by PCR of 50 μl, 25 cycles of 94 ° C 0:30, 0:45 55 ° C, 72 ° C 1:15, 72 ° C 8:00 and 4 ° C Hold Infinite.<br>
-
<ul>
+
 
-
<li><a href="Overview.htm">Overview<span class="flecha">&#9660;</span></a></li>
+
Following the amplification of the biobrick, a digestion was made for 8 hours at 37 ° C.<br>
-
           
+
Once obtained the promoters and GFP, each one was ligated to GFP reporter gene, by ligation of 16 hours at 16 ° C, with a final volume of 20 μL.<br>
-
            <li><a href="Light Response.htm">Light Response<span class="flecha">&#9660;</span></a></li>
+
Following the ligation of 16 hours, chemiocompetents TOP 10 cells were transformed by heat shock. At 12 hours of culture, the colonies were observed, the ones that were selected were those which showed green fluorescence under UV light. <br>
-
            <li><a href="Oxigen Response.htm">Oxigen Response<span class="flecha">&#9660;</span></a></li>
+
 
-
            <li><a href="Chassis.htm">Chassis<span class="flecha">&#9660;</span></a></li>
+
Miniprep was performed to extract plasmids of selected cells, as well as the vector J04450 cells. They were digested with the enzymes EcoRI and PstI for 16 hours.<br>
-
            <li><a href="Perspective.htm">Perspective<span class="flecha">&#9660;</span></a></li>
+
 
-
+
The bands were purified cutting the inserts constructions of promoters and the J04450 biobrick containing the chloramphenicol resistance.<br>
-
</ul>
+
 
-
</li>
+
Following this, a ligation of the promoter and the vector was made.<br>
-
<li>
+
 
-
<a href="">Results<span class="flecha">&#9660;</span></a>
+
From colonies which showed fluorescence, a final confirmation was performed by a digestion of the extracted DNA plasmid.</p>
-
<ul>
+
<p id="text2">Promoters Characterization</p>
-
<li><a href="Biobricks.htm">Biobricks<span class="flecha">&#9660;</span></a></li>
+
<p>With the selected colonies, an overnight culture was made in M9 media(minimal media supplemented with 0.2% CAA). After 12 hours the culture was transferred to a 96 well plate at a 1:10 dilution (20 μl of culture and 180 μL of fresh M9 medium).<br>
-
           
+
 
-
            <li><a href="Notebook.htm">Notebook<span class="flecha">&#9660;</span></a></li>
+
OD and fluorescence measurements of the selected colonies were performed at intervals of 30 minutes for 16 h.<br>
-
            <li><a href="Protocol.htm">Protocol<span class="flecha">&#9660;</span></a></li>
+
 
-
            <li><a href="Light Response.htm">Light Response<span class="flecha">&#9660;</span></a></li>
+
From the results the PopS were calculated (polymerases per second).
-
            <li><a href="Oxigen Response.htm">Oxigen Response<span class="flecha">&#9660;</span></a></li>
+
</p>
-
</ul>
+
  </div>
-
</li>
+
  <br><br>
-
+
    
-
<li>
+
 
-
<a href="#">IGEM Contribution<span class="flecha">&#9660;</span></a>
+
 
-
<ul>
+
  <div id="sidebar">
-
<li><a href="Construction.htm">Construction<span class="flecha">&#9660;</span></a></li>
+
    <div id="updates" class="orangebox">
-
           
+
      <h2>Promoter Selection</h2>
-
            <li><a href="Metods.htm">Metods<span class="flecha">&#9660;</span></a></li>
+
      <ul>
-
            <li><a href="Results.htm">Results<span class="flecha">&#9660;</span></a></li>
+
        <li><a href="Construction.htm" target="_parent">Construction</a></li>
-
+
        <li><a href="Metods.htm">Methods</a></li>
-
</ul>
+
      </ul>
-
</li>
+
    </div>
-
<li>
+
  </div>
-
<a href="Modelling.htm">Modelling<span class="flecha">&#9660;</span></a>
+
  <div style="clear: both;">&nbsp;</div>
-
<ul>
+
</div>
-
+
<!-- end #page -->
-
</ul>
+
<div id="piedepagina">
-
</li>    
+
  <p align="center"> <strong>Rhodofactory 2012</strong> </p>
-
   
+
<div id="sponsors">
-
    <li>
+
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/8/8a/Icytdf.png" alt="icytdf" width="90" height="82" /></div>
-
<a href="#">Team<span class="flecha">&#9660;</span></a>
+
</div>
-
<ul>
+
<div id="sponsors">
-
<li><a href="Members.htm">Members<span class="flecha">&#9660;</span></a></li>
+
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/6/67/Osli.png" alt="osli" width="88" height="46" /></div>
-
           
+
</div>
-
            <li><a href="Institute.htm">Institute<span class="flecha">&#9660;</span></a></li>
+
-
            <li><a href="Labs.htm">Labs<span class="flecha">&#9660;</span></a></li>
+
-
            <li><a href="Sponsor.htm">Sponsor<span class="flecha">&#9660;</span></a></li>
+
-
            <li><a href="Gallery.htm">Gallery<span class="flecha">&#9660;</span></a></li>
+
-
            <li><a href="Acknowledgments.htm">Acknowledgments<span class="flecha">&#9660;</span></a></li>
+
-
+
-
</ul>
+
-
</li>
+
-
    <li>
+
-
<a href="#">Outreach<span class="flecha">&#9660;</span></a>
+
-
<ul>
+
-
            <li><a href="Human Practice.htm">Human Practice<span class="flecha">&#9660;</span></a></li>
+
-
            <li><a href="Safety.htm">Safety<span class="flecha">&#9660;</span></a></li>
+
-
</ul>
+
-
</li>
+
-
   
+
-
   
+
-
</ul></td>
+
-
  <td><img src="images/spacer.gif" width="1" height="150" alt="" /></td>
+
-
  </tr>
+
-
  <tr>
+
-
  <td colspan="2" >  
+
-
 
+
-
  <div id="main"><a href="https://static.igem.org/mediawiki/2012/4/49/MATERIALS_AND_METHODS.pdf" target="_blank">Download PDF
+
-
    </a></div>
+
<div id="sponsors">
-
<div class="clearFloat"></div>
+
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/f/fb/Bio.png" alt="bio" width="80" height="97" /></div>
 +
</div>
 +
<div id="sponsors"><img src="https://static.igem.org/mediawiki/2012/d/d6/Fermentas.png" alt="fermentAS" width="82" height="45" /></div>
 +
<div id="sponsors">
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a3/Cinestav.png" alt="cinestav" width="74" height="91" /></div>
 +
</div>
 +
<div id="sponsors"><img src="https://static.igem.org/mediawiki/2012/9/9b/Genscript.png" alt="genscript" width="83" height="45" /></div>
 +
<div id="sponsors">
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/1/16/Unam.png" alt="unam" width="83" height="93" /></div>
 +
</div>
 +
<div id="sponsors"><img src="https://static.igem.org/mediawiki/2012/c/c9/Gto.png" alt="gto" width="84" height="46" /></div>
 +
    <div id="sponsors">
 +
      <div align="center"><img src="https://static.igem.org/mediawiki/2012/f/fa/Qimica.png" alt="quimica" width="80" height="72" /></div>
 +
    </div>
 +
<div id="sponsors"><img src="https://static.igem.org/mediawiki/2012/7/73/Valaner.png" alt="valaner" width="84" height="48" /></div>
 +
<div id="sponsors">
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/7/79/Ipn.png" alt="ipn" width="70" height="96" /></div>
 +
</div>
</div>
</div>
-
 
+
<!-- end #footer -->
-
  <img src="https://static.igem.org/mediawiki/2012/0/0f/Fig_4.jpg" width="19" /></td>
+
-
  <td><img src="images/spacer.gif" width="1" height="500" alt="" /></td>
+
-
  </tr>
+
-
</table>
+
</body>
</body>
</html>
</html>

Latest revision as of 02:00, 26 October 2012

Rho

Materials and Methods!

Biobicks

The following biobicks were recuperated from the 2009 and 2012 iGEM distribution plates.

figuram01

Circuits assembly

Plasmids were obtained by miniprep technique using the kit: Zyppy Plasmid Miniprep Kit and quantified by Nanodrop.

For the constitutive promoters (J23101, J23102, J23104, J23107, J23108, J23111 and J23115) the following enzyme digest were used: 8 hours at 37ºC:

figuram02


After enzyme digestion, a gel was made to confirm the released fragments and the band purification was performed using the kit: Gel DNA Recovery Kit.
The biobrick J54103 was extracted from the plate and amplified by PCR of 50 μl, 25 cycles of 94 ° C 0:30, 0:45 55 ° C, 72 ° C 1:15, 72 ° C 8:00 and 4 ° C Hold Infinite.
Following the amplification of the biobrick, a digestion was made for 8 hours at 37 ° C.
Once obtained the promoters and GFP, each one was ligated to GFP reporter gene, by ligation of 16 hours at 16 ° C, with a final volume of 20 μL.
Following the ligation of 16 hours, chemiocompetents TOP 10 cells were transformed by heat shock. At 12 hours of culture, the colonies were observed, the ones that were selected were those which showed green fluorescence under UV light.
Miniprep was performed to extract plasmids of selected cells, as well as the vector J04450 cells. They were digested with the enzymes EcoRI and PstI for 16 hours.
The bands were purified cutting the inserts constructions of promoters and the J04450 biobrick containing the chloramphenicol resistance.
Following this, a ligation of the promoter and the vector was made.
From colonies which showed fluorescence, a final confirmation was performed by a digestion of the extracted DNA plasmid.

Promoters Characterization

With the selected colonies, an overnight culture was made in M9 media(minimal media supplemented with 0.2% CAA). After 12 hours the culture was transferred to a 96 well plate at a 1:10 dilution (20 μl of culture and 180 μL of fresh M9 medium).
OD and fluorescence measurements of the selected colonies were performed at intervals of 30 minutes for 16 h.
From the results the PopS were calculated (polymerases per second).



 

Rhodofactory 2012

icytdf
osli
bio
fermentAS
cinestav
genscript
unam
gto
quimica
valaner
ipn

Retrieved from "http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Metods.htm"