Team:ULB-Brussels/Safety

From 2009.igem.org

(Difference between revisions)
(Page creation from Aline's .docx document)
(Header added)
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>iGEM Team:ULB-Brussels Wiki</title>
 +
<link rel="stylesheet" type="text/css" href="style.css" media="screen" />
 +
<link rel="stylesheet" type="text/css" href="menu.css" media="screen" />
 +
 +
<!-- Auto-Reload script for Dev -->
 +
<script language="javascript" type="text/javascript">
 +
<!--
 +
function timedRefresh(timeoutPeriod) {
 +
setTimeout("location.reload(true);",timeoutPeriod);
 +
}
 +
//  -->
 +
</script>
 +
        <style type="text/css">
 +
/*  Design for https://2009.igem.org/Team:ULB-Brussels
 +
 +
    Université Libre de Bruxelles
 +
*/
 +
 +
html body {
 +
width: 910px;
 +
font-family: Arial;
 +
margin: 0 auto;
 +
 +
}
 +
 +
html body div.header {
 +
margin: 0px;
 +
padding: 0px;
 +
margin-top: -21px; /* strange... */
 +
}
 +
 +
/* ===== banner ===== */
 +
 +
/* banner = large image on the top of the page, begind the title */
 +
 +
html body div.header div.banner {
 +
height: 171px;
 +
background-image: url(https://static.igem.org/mediawiki/2009/0/0a/Logoulb1.png);
 +
    background-repeat: no-repeat;
 +
}
 +
 +
html body div.header div.banner h1 {
 +
padding-top: 40px;
 +
   
 +
text-align: center;
 +
    text-decoration: none;
 +
    border: none;
 +
 +
color: #4E7DB7;
 +
font-size: 40px;
 +
}
 +
 +
html body div.header ul {
 +
    /* these are common to both 'menu' and 'submenu' */
 +
margin-top: 0px;
 +
margin-bottom: 0px;
 +
   
 +
list-style-type: none;
 +
    list-style-image: none;
 +
}
 +
 +
/* ===== menu ===== */
 +
 +
/* menu = horizontal bar containing the main links to other sections of the wiki */
 +
 +
html body div.header div.menu {
 +
    background-color: #5F91C1;
 +
color: white;
 +
   
 +
height: 30px;
 +
padding: 0px;
 +
}
 +
 +
html body div.header div.menu li {
 +
float: left;
 +
text-align: center;
 +
   
 +
    width: 120px;
 +
margin: 0px;
 +
margin-top: 10px;
 +
}
 +
 +
html body div.header div.menu li :hover {
 +
color: #5F7EB4;
 +
background-color: #FFFFFF;
 +
 +
margin: 0px;
 +
 +
padding-left: 15px;
 +
padding-right: 15px;
 +
padding-top: 4px;
 +
padding-bottom: 3px;
 +
}
 +
 +
/* class 'selection' refers to the 'selected' menu, in order to change it's color to white */
 +
html body div.header div.menu li a.selection {
 +
color: #5F7EB4;
 +
background-color: #FFFFFF;
 +
 +
margin: 0px;
 +
 +
padding-left: 15px;
 +
padding-right: 15px;
 +
padding-top: 4px;
 +
padding-bottom: 3px;
 +
}
 +
 +
html body div.header div.menu li a {
 +
color: white;
 +
text-decoration: none;
 +
}
 +
 +
/* ===== submenu ===== */
 +
 +
/* submenu = changing horizontal bar containing the main links to subsections of other sections of the wiki */
 +
 +
html body div.header div.submenu {
 +
/*background-color: #404040;*/
 +
background-color: white;
 +
height: 25px;
 +
 +
font-size: small;
 +
 +
border-bottom-style: solid;
 +
border-bottom-width: 1px;
 +
border-bottom-color: #AAAAAA;
 +
}
 +
 +
html body div.header div.submenu li {
 +
float: left;
 +
width: 120px;
 +
text-align: center;
 +
 +
margin: 0px;
 +
margin-top: 4px;
 +
}
 +
 +
html body div.header div.submenu li :hover {
 +
color: #5F7EB4;
 +
background-color: #FFFFFF;
 +
 +
margin: 0px;
 +
 +
padding-left: 15px;
 +
padding-right: 15px;
 +
padding-bottom: 3px;
 +
}
 +
 +
html body div.header div.submenu li a {
 +
color: #5F7EB4;
 +
text-decoration: none;
 +
}
 +
 +
/* ===== panel ===== */
 +
 +
/* panel = box containing mascotte, contdown and links to sponsors */
 +
 +
html body div.panel {
 +
    float: right;
 +
    border-left-style: solid;
 +
    border-left-width: 1px;
 +
    border-left-color: #AAAAAA;
 +
   
 +
    padding-left: 10px;
 +
    margin-left: 10px;
 +
    margin-top: 20px;
 +
   
 +
    width: 250px;
 +
    font-size: small;
 +
}
 +
 +
/* appearance of the links */
 +
html body div.panel a {
 +
    text-decoration: none;
 +
    font-weight: bold;
 +
    color: #3E6DA4;
 +
}
 +
 +
html body div.panel a:hover {
 +
    color: #F5B455;
 +
}
 +
 +
/* == countdown == */
 +
 +
html body div.panel div.countdown {
 +
    /*margin-top: 20px;
 +
    margin-bottom: 20px;
 +
    height: 30px;*/
 +
    margin: 20px;
 +
    padding: 20px;
 +
}
 +
 +
.countdown font {
 +
    padding: 20px;
 +
    margin: 20px;
 +
    color: #5F7EB4;
 +
}
 +
 +
/* == sponsors == */
 +
 +
html body div.panel div.sponsor {
 +
    margin-bottom: 10px;
 +
    min-height: 50px;
 +
}
 +
 +
/* appearance of the sponsor's logos */
 +
html body div.panel div.sponsor img {
 +
    width: 50px;
 +
    height: 50px;
 +
    border-style: none;
 +
    float: left;
 +
    margin-right: 7px;
 +
}
 +
 +
/* ===== main ===== */
 +
 +
html body div.main {
 +
font-size: small;
 +
padding-left: 10px;
 +
padding-right: 10px;
 +
    width: 625px;
 +
    text-align: justify;
 +
}
 +
 +
html body div.main p.abstract {
 +
font-weight: bold;
 +
}
 +
 +
html body div.main div.welcome img {
 +
    width: 616px;
 +
    height: 140px;
 +
    margin-top: 10px;
 +
    border-style: none;
 +
}
 +
 +
html body div.footer {
 +
}
 +
 +
        </style>
 +
       
 +
</head>
 +
 +
    <!-- *** Uncomment next line to enable autorefresh (developper mode) *** -->
 +
<!-- <body onload="JavaScript:timedRefresh(5000);"> -->
 +
<!-- <body> -->
 +
    <body>
 +
        <!--
 +
            header : box on the top of the page containing the banner and links to sections and subsections of
 +
            the wiki.
 +
        -->
 +
<div class="header">
 +
           
 +
<div class="banner">
 +
<h1></h1>
 +
</div>
 +
<div class="menu">
 +
<ul>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels">Home</a></li>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels/Team">Team</a></li>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels/Project">Project</a></li>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels/Parts">Parts</a></li>
 +
<li><a class="mainlink selection" href="/Team:ULB-Brussels/Safety">Safety</a></li>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels/Notebook">Notebook</a></li>
 +
<li><a class="mainlink" href="/Team:ULB-Brussels/Sponsors">Sponsors</a></li>
 +
</ul>
 +
</div>
 +
<div class="submenu">
 +
<ul>
 +
<!-- <li><a href="#Contact">Contact</a></li>
 +
<li><a href="#Advisors">Advisors</a></li>
 +
<li><a href="#Students">Students</a></li>
 +
-->
 +
</ul>
 +
</div>
 +
</div>
 +
       
 +
        <!--
 +
            panel : box on the right containing a countdown, links to the sponsors
 +
 +
            [ REMOVED ]
 +
        -->
 +
 +
  </div> 
 +
 +
<div class="footer">
 +
</div>
 +
 +
</body>
 +
</html>
 +
 +
 +
= GluColi and a free-antibiotics alternative =
 +
 +
In this work, we were aware of some ethical issues, in particular the use of antibiotics.
In this work, we were aware of some ethical issues, in particular the use of antibiotics.
Antibiotics are used to select all types of plasmidic vectors in bacteria which is a significant concern in industrial cultures producing recombinant proteins or DNA.
Antibiotics are used to select all types of plasmidic vectors in bacteria which is a significant concern in industrial cultures producing recombinant proteins or DNA.
Line 8: Line 306:
In the Staby<sup>TM</sup> system, the antidote gene (ccdA) is introduced in the plasmid DNA under the control of a weak constitutive promoter: the mob gene promoter, which originates from a broad host range plasmid (pBHR1). On the other hand, the toxic gene (ccdB) is introduced in the E. coli chromosome of the bacteria. Expression of the poison gene is under the control of a promoter strongly repressed in the presence of the plasmid. Practically, 100% of the bacteria will carry the vector. If they lose the vector, they will not obtain a growth advantage, but will die. Upon induction, all the bacteria will start producing the recombinant protein. It will lead to higher yields of the recombinant protein and less background caused by unwanted proteins. Therefore, higher plasmid stability means higher protein of interest production.
In the Staby<sup>TM</sup> system, the antidote gene (ccdA) is introduced in the plasmid DNA under the control of a weak constitutive promoter: the mob gene promoter, which originates from a broad host range plasmid (pBHR1). On the other hand, the toxic gene (ccdB) is introduced in the E. coli chromosome of the bacteria. Expression of the poison gene is under the control of a promoter strongly repressed in the presence of the plasmid. Practically, 100% of the bacteria will carry the vector. If they lose the vector, they will not obtain a growth advantage, but will die. Upon induction, all the bacteria will start producing the recombinant protein. It will lead to higher yields of the recombinant protein and less background caused by unwanted proteins. Therefore, higher plasmid stability means higher protein of interest production.
-
[[Image:Staby.png|frame|center|[http://www.delphigenetics.com/ Delphi Genetics]'s Staby system]]
+
[[Image:Staby.png|frame|center|Staby<sup>TM</sup> system from [http://www.delphigenetics.com/ Delphi Genetics]]]
We think it could be relevant to implement this antibiotic free expression strategy in our project. This is the reason why we designed 2 bricks containing the ccdA gene. In order to carry out transformation, bacteria should be ordered with the ccdB gene in their genome from Delphi Genetics.
We think it could be relevant to implement this antibiotic free expression strategy in our project. This is the reason why we designed 2 bricks containing the ccdA gene. In order to carry out transformation, bacteria should be ordered with the ccdB gene in their genome from Delphi Genetics.

Revision as of 11:32, 21 October 2009

iGEM Team:ULB-Brussels Wiki


GluColi and a free-antibiotics alternative

In this work, we were aware of some ethical issues, in particular the use of antibiotics. Antibiotics are used to select all types of plasmidic vectors in bacteria which is a significant concern in industrial cultures producing recombinant proteins or DNA. As plasmid-free cells growing faster than plasmid-carrying cells, the yield and the production reproducibility of recombinant molecules are significantly lowered in the absence of plasmid selection by the appropriate antibiotic. To overcome the plasmid instability, many antibiotic resistance genes are used as selectable markers in fermentation processes. Antibiotics are expensive and they pose safety problems: they contaminate the production product. The Belgian company Delphi Genetics has developed a strategy called “StabyTM system” which relies on the use of poison-antidote genes.

The StabyTM system is based on the poison-antidote ccd module. Poison-antidote modules are found in natural plasmid in which they serve to the plasmid maintenance. The CcdB protein (poison) is cytotoxic and poisons DNA-gyrase complexes. Expression of this gene in the absence of its cognate ccdA antidote leads the death of the bacteria. The product of the ccdA gene (antidote) antagonizes this toxic activity by forming a poison-antidote protein complex. If a plasmid carrying the ccd module is lost at cell division, the plasmid-free bacteria contain poison and antidote proteins in their cytoplasm. Since the antidote is unstable and degraded by a host protease, the poison will be free and able to poison DNA-gyrase complexes. This will lead eventually to cell death.

In the StabyTM system, the antidote gene (ccdA) is introduced in the plasmid DNA under the control of a weak constitutive promoter: the mob gene promoter, which originates from a broad host range plasmid (pBHR1). On the other hand, the toxic gene (ccdB) is introduced in the E. coli chromosome of the bacteria. Expression of the poison gene is under the control of a promoter strongly repressed in the presence of the plasmid. Practically, 100% of the bacteria will carry the vector. If they lose the vector, they will not obtain a growth advantage, but will die. Upon induction, all the bacteria will start producing the recombinant protein. It will lead to higher yields of the recombinant protein and less background caused by unwanted proteins. Therefore, higher plasmid stability means higher protein of interest production.

StabyTM system from Delphi Genetics

We think it could be relevant to implement this antibiotic free expression strategy in our project. This is the reason why we designed 2 bricks containing the ccdA gene. In order to carry out transformation, bacteria should be ordered with the ccdB gene in their genome from Delphi Genetics.

Retrieved from "http://2009.igem.org/Team:ULB-Brussels/Safety"