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

From 2012.igem.org

(Difference between revisions)
(Blanked the page)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
 +
<html xmlns="http://www.w3.org/1999/xhtml">
 +
<head>
 +
<meta http-equiv="content-type" content="text/html; charset=utf-8" />
 +
<title>Rho</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;
 +
}
 +
 +
* {
 +
margin: 0;
 +
padding: 0;
 +
}
 +
 +
body {
 +
background: #FFFFFF url(https://static.igem.org/mediawiki/2012/f/f3/Fondo_bod.jpg) repeat-x;
 +
text-align: justify;
 +
font-family: Georgia, "Times New Roman", Times, serif;
 +
font-size: 12px;
 +
color: #000000;
 +
}
 +
 +
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;
 +
}
 +
 +
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: 60%;
 +
color: #FFFFFF;
 +
}
 +
 +
#sidebar ul {
 +
margin: 0;
 +
padding: 15px;
 +
list-style: none;
 +
}
 +
 +
#sidebar li {
 +
              font-size: 12px;
 +
  text-align:left;
 +
}
 +
 +
#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 */
 +
 +
#piep {
 +
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;
 +
 +
      }
 +
#text2
 +
{
 +
color: #333333;
 +
font-size: 152%;
 +
margin-bottom: 20px;
 +
font-weight: normal;
 +
}
 +
#refe
 +
{
 +
color: #000000;
 +
font-size:9;
 +
margin-bottom: 20px;
 +
font-weight: normal;
 +
}
 +
.Estilo2 {color: #FDFDFD; font-weight: bold; }
 +
.Estilo3 {
 +
color: #FFFFFF;
 +
font-weight: bold;
 +
}
 +
 +
.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>
 +
<body>
 +
<div id="menu">
 +
<ul>
 +
<li><a href="home.htm">Home</a></li>
 +
<li><a href="rhodofactory.htm">Rhodofactory</a></li>
 +
<li><a href="promoterselection.htm">Promoter selection </a></li>
 +
<li class="active"><a href="results.htm">results </a></li>
 +
<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>Light and oxygen response!</h1>
 +
<p id="text2">AppA/PpsR Regulation System</p>
 +
<p>In this repressor/antirepressor system, under low oxygen tension, the GFP
 +
expression is possible because the repressor PpsR is forming a complex with the
 +
antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a
 +
conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See
 +
Rhodofactory section for a complete explanation).<br><br>
 +
 +
 +
We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light &
 +
Oxygen Control System, each one has GFP as a reporter gene and the functionality was
 +
related to the fluorescence detection.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/4/40/Oxy01.jpg" alt="oxy01" width="561" height="241">
 +
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the
 +
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.palustris.</em></p>
 +
<img src="https://static.igem.org/mediawiki/2012/1/1c/Oxy02.jpg" width="563" height="183">
 +
<p>Figure 2. This BioBrick will show if our complete system is functional because probably
 +
we need a synthetic system to promote GFP expression by binding its target sequence
 +
(dependent promoter) in <em>R. palustris.</em></p>
 +
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur
 +
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R.palustris</em>,
 +
by biparental and triparental conjugation.</p>
 +
<p>The measurement approach was:
 +
<li>Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.</li>
 +
<li>Flow Cytometry: To have a quantitative GFP detection, we calculated the percentage of
 +
bacterial population expressing GFP (GFP+) in 1000 bacteria.</li></p><br>
 +
 +
<p>We used 3 environmental growing conditions:
 +
<li>Aerobic/Darkness</li>
 +
<li>Anaerobic/Light</li>
 +
<li>Anaeroibic/darkness</li>
 +
</p><br>
 +
 +
<p>For all data results, we considered a negative control: <em>R. sphaeroides</em> and
 +
<em>R.palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f0/Oxygenres01.jpg" width="563" height="452">
 +
<img src="https://static.igem.org/mediawiki/2012/c/cc/Oxygenres02.jpg" width="563" height="452">
 +
<p>Figure 3. Percentage of bacterial population expressing GFP..<br>
 +
</p>
 +
  <img src="https://static.igem.org/mediawiki/2012/9/9b/Oxy03.jpg" width="561" height="465"></div>
 +
<div align="center">
 +
</div>
 +
<p>Figure 4. Images obtained by fluorescence microscopy, where our systems
 +
were functional in the expected conditions.</p>
 +
<p id="text2">Discussion</p>
 +
<p>In <em>R. sphaeroides</em>, the best functionality of our system was in aerobic and darknesss
 +
condition, it was not the expected result, but probably the activation is because to the
 +
incomplete repression of the system because we were overexpressing the constitutive
 +
proteins. Altought, we obtained GFP expression with a lower level, in the expected
 +
condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.<br>
 +
<br>
 +
 +
In <em>R.palustris</em>, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system. </p>
 +
 +
<p id="text2">Conclusion</p>
 +
<p>Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic
 +
bacteria <em>R.palustris</em> and <em>R. sphaeroides.</em> The best condicion was aerobic/darknes and anaerobic light respectively.
 +
 +
 +
This is a functional system for controlling genetic expression with Light and Oxygen signals.</p>
 +
 +
</div>
 +
 +
<div id="sidebar">
 +
  <div id="updates" class="orangebox">
 +
 +
    <h2>Results</h2>
 +
<ul>
 +
<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li>
 +
<li><a href="Lightandoxre.htm">Light and oxygen response</a></li>
 +
<li><a href="Notebook.htm">Notebook</a></li>
 +
<li><a href="oxigenresponse.htm">Oxygen response</a></li>
 +
<li><a href="Protocol.htm">Protocols</a></li>
 +
</ul>
 +
</div>
 +
  </div>  
 +
  <div style="clear: both;">&nbsp;</div>
 +
</div>
 +
<!-- end #page -->
 +
<div id="piep">
 +
<p align="center"> <strong>Rhodofactory 2012</strong> </p>
 +
<div id="sponsors">
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/8/8a/Icytdf.png" alt="icytdf" width="90" height="82" /></div>
 +
</div>
 +
<div id="sponsors">
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2012/6/67/Osli.png" alt="osli" width="88" height="46" /></div>
 +
</div>
 +
 +
<div id="sponsors">
 +
  <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>
 +
<!-- end #footer -->
 +
</body>
 +
</html>

Latest revision as of 22:59, 26 October 2012

Rho

Light and oxygen response!

AppA/PpsR Regulation System

In this repressor/antirepressor system, under low oxygen tension, the GFP expression is possible because the repressor PpsR is forming a complex with the antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See Rhodofactory section for a complete explanation).

We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light & Oxygen Control System, each one has GFP as a reporter gene and the functionality was related to the fluorescence detection.

oxy01

Figure 1. This BioBrick will show if our dependent promoter is functional, using the constitutive (or natural) system from R. sphaeroides or the orthologue system from R.palustris.

Figure 2. This BioBrick will show if our complete system is functional because probably we need a synthetic system to promote GFP expression by binding its target sequence (dependent promoter) in R. palustris.

Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R.palustris, by biparental and triparental conjugation.

The measurement approach was:

  • Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.
  • Flow Cytometry: To have a quantitative GFP detection, we calculated the percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.


    • We used 3 environmental growing conditions:

  • Aerobic/Darkness
  • Anaerobic/Light
  • Anaeroibic/darkness


    • For all data results, we considered a negative control: R. sphaeroides and R.palustris, conjugated bacteria with pRK415 vector without BioBrick.

    Figure 3. Percentage of bacterial population expressing GFP..

    Figure 4. Images obtained by fluorescence microscopy, where our systems were functional in the expected conditions.

    Discussion

    In R. sphaeroides, the best functionality of our system was in aerobic and darknesss condition, it was not the expected result, but probably the activation is because to the incomplete repression of the system because we were overexpressing the constitutive proteins. Altought, we obtained GFP expression with a lower level, in the expected condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.

    In R.palustris, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system.

    Conclusion

    Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic bacteria R.palustris and R. sphaeroides. The best condicion was aerobic/darknes and anaerobic light respectively. This is a functional system for controlling genetic expression with Light and Oxygen signals.

     

    Rhodofactory 2012

    icytdf
    osli
    bio
    fermentAS
    cinestav
    genscript
    unam
    gto
    quimica
    valaner
    ipn

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