Team:DTU-Denmark-2/Project/introduction

From 2011.igem.org

(Difference between revisions)
Line 1: Line 1:
{{:Team:DTU-Denmark-2/Header}}
{{:Team:DTU-Denmark-2/Header}}
-
 
<html>
<html>
 +
<head>
 +
 +
<style type="text/css">
 +
 +
A {text-decoration:none}
 +
A:link {color:  #990000;text-decoration: none }
 +
A:active {color:  #990000;text-decoration: none }
 +
A:visited {color:  #990000 ;text-decoration: none }
 +
A:hover {text-decoration: underline}
 +
 +
 +
 
 +
  div.text {
 +
    padding-right: 20px;
 +
    padding-left: 50px;
 +
    padding-top:20px;
 +
    padding-bottom:10px;
 +
}
 +
 +
table {
 +
  border-collapse:collapse;
 +
}
 +
table,th, td {
 +
}
 +
 +
#menu {
 +
  width: 200px;
 +
  position: relative;
 +
  top: 0px;
 +
}
 +
a.h1 {
 +
  font-size: 12pt;
 +
  font-weight: bold;
 +
}
 +
 +
a.h2 {
 +
  font-size: 9pt;
 +
  margin-left: 10px;
 +
}
 +
 +
</style>
 +
 +
</head>
<body>
<body>
 +
 +
<div class="text">
 +
<font color=" #990000" face="arial" size="6">
<br><br>
<br><br>
-
<font color="#990000" face="arial" size="6" >
+
<b>Introduction</b><br> <br>  
-
<p align="left">
+
</font>
-
<b>The world calls for a better Assembly System</b><br>
+
-
</p>
+
-
  </font>
+
-
<p align="justify">
+
-
<b>Synthetic Biology</b><br>
+
 
-
</p>
+
 
 +
</div>
 +
 
 +
<table cellpadding=5px>
 +
<th>
 +
<td width="500px" height="100%" valign="top" valign="center" >
 +
<br>
 +
 
 +
 
 +
<div id="menu">
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#Synthetic Biology" class="h1"><b>1</b> Synthetic Biology</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#The world calls for a better Assembly System" class="h1"> <b>2</b> The world calls for a better Assembly System</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#USER cloning" class="h1"><b>3</b> USER cloning</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#Outline of the Plug 'n' Play with DNA idea" class="h1"><b>4</b> Outline of the Plug 'n' Play with DNA idea</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#The mission of Plug 'n' Play with DNA"
 +
class="h1"><b>5</b> The mission of Plug 'n' Play with DNA</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#Application area" class="h1"><b>6</b> Application area</a><br><br>
 +
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/Introduction#References" class="h1"><b>7</b> References</a><br><br>
 +
 
 +
</div>
 +
 
 +
</td>
 +
 
 +
<td width="75%" height="100%" valign="top">
 +
 
 +
 
 +
 
 +
 
 +
<br>
 +
 
 +
<a name="Synthetic Biology"></a><h1><b>Synthetic Biology</b></h1>
 +
<p align="justify">
Synthetic Biology is the engineering of Biology
Synthetic Biology is the engineering of Biology
Line 34: Line 106:
-
<br><br>
+
 
-
<font color="#990000" face="arial" size="6" >
+
<a name="The world calls for a better Assembly System"></a><h1><b>The world calls for a better Assembly System</b></h1>
-
<p align="left">
+
 
-
<b>The world calls for a better Assembly System</b><br>
+
-
</p>
+
-
  </font>
+
<p align="justify">
<p align="justify">
The Registry of Standard Biological Parts and iGEM make use of the Standard Assembly of BioBricks formulated by Tom Knight (kilde. The Standard Assembly make use of restrictionsites for the four restriction enzymes EcoRI, XbaI, SpeI , and PstI. The BioBricks have to hold the restrictionsites for the two enzymes EcoRI and XbaI prefix and suffix the restrictionsites for the two enzymes SpeI and PstI. In order to have assembly correct the BioBrik can't contain any of these four restrictionsites within the BioBrick. However, if any of this four restriction sites are present, they will have to be eliminated by alterations like site-directed mutagenesis, which can be both time consuming and cause unwanted alterations. In developing new BioBricks from natural sources and higher organism than eukaryotes the illegal restricktion sites can be of a problem. Furthermore, when assembling BioBricks with the Standard Assembly System scars occur, which makes it impossible to create fusion proteins. Additionally, the Standard Assembly of BioBricks is limited in that it is only possible to put two BioBricks together at a time.
The Registry of Standard Biological Parts and iGEM make use of the Standard Assembly of BioBricks formulated by Tom Knight (kilde. The Standard Assembly make use of restrictionsites for the four restriction enzymes EcoRI, XbaI, SpeI , and PstI. The BioBricks have to hold the restrictionsites for the two enzymes EcoRI and XbaI prefix and suffix the restrictionsites for the two enzymes SpeI and PstI. In order to have assembly correct the BioBrik can't contain any of these four restrictionsites within the BioBrick. However, if any of this four restriction sites are present, they will have to be eliminated by alterations like site-directed mutagenesis, which can be both time consuming and cause unwanted alterations. In developing new BioBricks from natural sources and higher organism than eukaryotes the illegal restricktion sites can be of a problem. Furthermore, when assembling BioBricks with the Standard Assembly System scars occur, which makes it impossible to create fusion proteins. Additionally, the Standard Assembly of BioBricks is limited in that it is only possible to put two BioBricks together at a time.
Line 50: Line 119:
<br><br>
<br><br>
-
<font color="#990000" face="arial" size="6" >
+
<a name="USER cloning"></a><h1><b>USER cloning</b></h1>
-
<p align="left">
+
-
<b>USER cloning</b><br>
+
-
</p>
+
-
  </font>
+
<br>
<br>
<p align="left">
<p align="left">
Line 62: Line 127:
</p>
</p>
-
 
+
<a name="Outline of the Plug 'n' Play with DNA idea"></a><h1><b>Outline of the Plug 'n' Play with DNA idea</b></h1>
-
<font color="#990000" face="arial" size="6" >
+
-
<p align="left">
+
-
<b>Outline of the Plug 'n' Play with DNA idea </b><br>
+
-
</p>
+
-
  </font>
+
<center><img src="https://static.igem.org/mediawiki/2011/d/d0/Introduction_ny.png" height="400px"> </img></center>
<center><img src="https://static.igem.org/mediawiki/2011/d/d0/Introduction_ny.png" height="400px"> </img></center>
Line 73: Line 134:
-
<font color="#990000" face="arial" size="6" >
+
 
-
<p align="left">
+
<a name="The mission of Plug 'n' Play with DNA"></a><h1><b>The mission of Plug 'n' Play with DNA</b></h1>
-
<b>Plug 'n' Play with DNA mission</b><br>
+
-
</p>
+
-
  </font>
+
<p align="left">
<p align="left">
Line 83: Line 141:
</p>
</p>
-
<font color="#990000" face="arial" size="6" >
 
-
<p align="left">
 
-
<b>Application of the Plug 'n' Play Assembly standard</b><br>
 
-
</p>
 
-
  </font>
 
 +
<a name="Application area"></a><h1><b>Application area</b></h1>
<br>
<br>
-
<font color="#990000" face="arial" size="4" >
+
<br>
-
<p align="left">
+
 
-
<b>References</b><br>
+
<a name="References"></a><h1><b>References</b></h1>
-
</p>
+
 
-
  </font>
+
<p align="justify">
<p align="justify">
(1) Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.
(1) Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.
Line 114: Line 167:
<br>
<br>
<br>
<br>
 +
 +
 +
 +
</ul>
 +
</td>
 +
</tr>
 +
</table>
 +
<div id="EndOfPage"></div>
 +
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js" charset="utf-8"></script>
 +
<script type="text/javascript" charset="utf-8">
 +
$(function(){
 +
//  $('#test').text($(this).scrollTop());
 +
  var menu = $('#menu')
 +
  var pos = menu.position().top;
 +
  var offset = 20;
 +
 +
  $(window).scroll(function(){
 +
    $('#test').text(menu.position().top);
 +
    if(($(this).scrollTop() + menu.height() + offset) > $('#EndOfPage').position().top) {
 +
      menu.css("position", "absolute");
 +
      menu.css("top", ($('#EndOfPage').position().top - menu.height()) + "px");
 +
    }else{
 +
 +
      if(($(this).scrollTop() + offset) > pos) {
 +
        menu.css("position", "fixed");
 +
        menu.css("top", offset + "px");
 +
      }else{
 +
        menu.css("position", "relative");
 +
        menu.css("top", "0px");
 +
      }
 +
    }
 +
 +
 +
  });
 +
});
 +
</script>
 +
 +
 +
</body>
</body>
</html>
</html>

Revision as of 15:55, 18 September 2011




Introduction


Synthetic Biology

Synthetic Biology is the engineering of Biology

[1] "Synthetic Biology. Applying Engineering to Biology". Report of a NEST High-Level Expert Group. European Commission, Directorate-General for Research. Available online at http://www.synbiosafe.eu/uploads///pdf /EU-highlevel-syntheticbiology.pdf
[2] Endy, D. Foundations for engineering biology. Nature 438: 449-453 ()2005)
[3] Endy, D. Deese I. Adventures in synthetic biology (comic).Nature 438: 449-453 (2005)Available online at nature.com
[4] http://www.partsregistry.org/Assembly:Standard_assembly (Website, accessed 10/15/2009)

Hvad er? Parta (DNA, genes and proteins) devices (a number of parts combined to process inputs to a functional output) modules (a set of interconnected devices that performs complex tasks) The majority of the teams uses E. coli as chassis. Concerning eukaryotes within the iGEM competition most projects focused on yeast as chassis. There are very few teams which actually worked with mammalian cells. - Heidelberg 2009, Slovenia 2006 mammalian celle, fungi - kan ikke lige huske? Hvorfor er mammale og fungi så vigitige? (Mammale - medicink brug og tearpuetiske lægmiddeler, fungi producere nogle af de mest advancered molekyler til medicink brug, samt celle fabriker for en stor udvalg af komercielle varer som citrc acid, enzymer, lactic acid, farvestoffer.

The world calls for a better Assembly System

The Registry of Standard Biological Parts and iGEM make use of the Standard Assembly of BioBricks formulated by Tom Knight (kilde. The Standard Assembly make use of restrictionsites for the four restriction enzymes EcoRI, XbaI, SpeI , and PstI. The BioBricks have to hold the restrictionsites for the two enzymes EcoRI and XbaI prefix and suffix the restrictionsites for the two enzymes SpeI and PstI. In order to have assembly correct the BioBrik can't contain any of these four restrictionsites within the BioBrick. However, if any of this four restriction sites are present, they will have to be eliminated by alterations like site-directed mutagenesis, which can be both time consuming and cause unwanted alterations. In developing new BioBricks from natural sources and higher organism than eukaryotes the illegal restricktion sites can be of a problem. Furthermore, when assembling BioBricks with the Standard Assembly System scars occur, which makes it impossible to create fusion proteins. Additionally, the Standard Assembly of BioBricks is limited in that it is only possible to put two BioBricks together at a time.



All in all, the iGEM competition and the fast growing field of synthethic biology calls for a simpler, faster and more efficient assembly system that are easily applied to both bacteria, fungi, and mammalian cells.



USER cloning


In early 1990s, the uracil excision-based (USER) cloning was invented as a ligation-independent cloning technique that could substitute the conventional assembly systems that made use of restriction enzymes and ligase. In 2003 New England Biolabs (NEB) introduced the USER Friendly Cloning Kit. Although NEBs USER kit was simple and efficient, it was not compatible with proofreading polymerases that stalled when encountering a uracil base in the DNA template (2. New England Biolabs, 2004). This made the USER Friendly Kit unattractive, although the concept was brilliant. In recent years, proofreading polymerases have been developed that are compatible with the concept of USER cloning, since they can read through uracil (1. Nour-Eldin HH etc)..

The USER method applies long complementary overhangs on the PCR product(s) as well as on the destination vector. The overhangs on the PCR product are custom made, between 7-15 nucleotides long and deoxy uridine nucleotides substitute selected deoxy thymidine nucleotides. The PCR products containing the customized overhangs are treated with the USER enzyme, which is a mix of DNA glycosidase and DNA glycosylase-lyase endo VIII. This treatment results in release of the DNA sequence upstream the deoxy uridine nucleotide and the resulting exposed overhangs can anneal to each other to form a stable hybridization product. This product can now be transformed directly into E.coli without prior ligation (1,2). In order to avoid template carry-over after PCR, the PCR product is usually treated with the restriction enzyme DpnI. DpnI cleaves only when its recognition site is methylated. Unmethylated PCR-derived DNA will be left intact (1)……

Outline of the Plug 'n' Play with DNA idea




The mission of Plug 'n' Play with DNA

DTU-Denmark-2 introduces a customized system called "Plug ‘n’ Play with DNA", where certain categories of biological parts can be gathered. This means that the parts in the form of pre-produced PCR-products are directly mixed with a backbone vector, which makes assembly of an expression vector possible in less than one day. All the parts in the form of PCR-products will be distributed in microtiter plates directly ready for cloning. Furthermore, the “Plug ’n’ Play” kit will contain a back-up plasmid of all parts to ensure amplification from a mutation free template if needed. The simple and easy use of the system will be demonstrated by developing a reporter targeting system for the filamentous fungi of Aspergilli as well as for mammalian cells. In collaboration with the Copenhagen iGEM team will we demonstrate the easy use of the system for bacteria E. coli.

Application area



References

(1) Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.

(2) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).

 




 

Retrieved from "http://2011.igem.org/Team:DTU-Denmark-2/Project/introduction"