Team:DTU-Denmark/BBrick Characterisation

From 2010.igem.org

(Difference between revisions)
 
(9 intermediate revisions not shown)
Line 1: Line 1:
-
__NOTOC__
 
-
[[image:wiki banner_967px.png]]
 
-
 
<html>
<html>
 +
<img src="https://static.igem.org/mediawiki/2010/6/6b/Wiki_banner_967px.png"  align="center"> </img>
<head>
<head>
<title>Welcome to the DTU iGEM wiki!</title>
<title>Welcome to the DTU iGEM wiki!</title>
Line 43: Line 41:
.links tr {
.links tr {
-
  background-color:#660066;
+
  background-color:#990000;
  height:30px;
  height:30px;
}
}
Line 67: Line 65:
.links a:hover {
.links a:hover {
-
background-color: #660066;
+
background-color: #999999;
}
}
Line 152: Line 150:
<tr>
<tr>
<td width="163px" height="100%" valign="top">
<td width="163px" height="100%" valign="top">
 +
  <font color="#990000" face="arial" size="3">
 +
<ul type="circle">
 +
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Basics">Basics</a></li><br>
 +
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems">Regulatory Systems</a></li><br>
 +
<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li><br>
 +
<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><br>
 +
<li ><a href="https://2010.igem.org/Team:DTU-Denmark/BBrick_Characterisation">Results</a></li><br>
 +
</ul>
</td>
</td>
<td>
<td>
-
<h2>Introduction</h2>
+
<h1>Introduction</h1>
-
<p align="justify">Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (????  By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts.
+
<p align="justify">In order to optimize the lab work, we split up the work so that we could have two lab teams working in parallel to design different parts of the switch. We split up the lab work so we had:<br>
-
In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.</p>
+
<ul>
-
<h2>Anti-Terminator</h2>
+
<li>Team 1: <b>The Repressor - Anti-Repressor Team</b></li>
-
<p align="justify">Introduction to this part</p>
+
<li>Team 2: <b>The Terminator - Anti-Terminator Team</b></li>
-
<h3>Selecting subparts</h3>
+
</ul>
-
<p align="justify">Why were these subparts chosen ?<br>
+
The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:<br>
-
AIM</p>
+
<p align="center"><img scr=""></img></p>
-
<h3>Design and experimental setup</h3>
+
The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:<br>
-
<p align="justify">presentation - Figure of setup and explanation</p>
+
<p align="center"><img scr=""></img></p>
-
<h3>Materials and methods</h3>
+
</p>
-
<p align="justify">HOW ? what plasmids and why, what measurering method and why?<br>
+
<h1>Repressor Group</h1>
-
refer to the notebook page with protocols - and actual info from lab.<br>
+
<p align="justify">The repressor group will be assembling the constructs step-by-step:</p>
-
<b>mRNA-stability</b><br>
+
<h3>Step 1</h3>
-
when introducing non-coding sequences problems will acour  with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)<br>
+
<p align="justify">The construction of a plasmid containing the divergent promoters is the first step, the effect of this will be the uninhibited expression of GFP as illustrated by the green colonies observed in Figure 4.</p>
-
<h3>Results</h3>
+
<p align="center"><img scr=""></img></p>
-
<p align="justify">comments to the results and reference to the BB pages with info and results.</p><br>
+
<font size="1.5">
-
 
+
<p align="justify"><b>Figure 3</b>: The initial plasmid constructed is illustrated. The divergent promoters have been inserted into a plasmid and transformed into the electro-competent <i>E.coli</i> cells.</p>
-
<h2>Repressor function</h2>
+
<p align="center"><img scr=""></img></p>
-
<p align="justify">Introduction to this part</p>
+
<p align="justify"><b>Figure 3</b>: The success of the plasmid construction and transformation is illustrated by the fluorescent green colonies seen on the LB-agar plates.</p>
-
<h3>Selecting subparts</h3>
+
</font>
-
<p align="justify">Why were these subparts chosen ?<br>
+
<h3>Step 2</h3>
-
AIM</p>
+
<p align="center"><img src="https://static.igem.org/mediawiki/2010/e/ef/DTU_BB_Repressor1.png" width="570px"  align="center"> </img></p>
-
<h3>Design and experimental setup</h3>
+
<font size="1.5">
-
<p align="justify">presentation - Figure of setup and explanation</p>
+
<p align="justify"><b>Figure 4</b>: The construction of a plasmid containing the Repressor protein (GogR or GtgR) expressed from the pRM promoter is shown. The continually expressed repressor protein will then inhibit the pR promoter and no GFP will be expressed.</p>
-
<h3>Materials and methods</h3>
+
</font>
-
<p align="justify">HOW ? what plasmids and why, what measurering method and why?<br>
+
<h3>Step 3</h3>
-
refer to the notebook page with protocols - and actual info from lab.<br>
+
<font size="1.5">
-
<h3>Results</h3>
+
<p align="center"><img src="https://static.igem.org/mediawiki/2010/7/7c/DTU_BB_Repressor2.png" width="570px"  align="center"> </img></p>
-
<p align="justify">comments to the results and reference to the BB pages with info and results.</p><br>
+
<p align="justify"><b>Figure 5</b>: The independent plasmid is constructed is shown. This plasmid contains the gene encoding the anti-repressor is found downstream of the promoter induced by arabinose, pBAD.</p>
-
 
+
</font>
-
<h2>Synthetic promoter library (SPL)</h2>
+
<h3>Results Simulation</h3>
-
How does it work, examples, what have it been used to characterize?<br>
+
<font size="1.5">
-
how do you construct it? Figures and illustrations to explain.<br>
+
<p align="center"><img src="https://static.igem.org/mediawiki/2010/3/34/DTU_BB_Repressor3_graph.png" width="570px"  align="center"> </img></p>
-
Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.<br></p>
+
<p align="justify"><b>Figure 7</b>: The graphs illustrated are a simulation of the expected results from <b>Construct 2</b> and <b>Construct 3</b>. The expected results from <b>Construct 2</b> would be a baseline expression of GFP, as the promoter would continually be repressed. With <b>Construct 3</b>, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.</p>
 +
</font>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2010/a/a0/DTU_BB_AntiT1.png" width="570px"  align="center"> </img></p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2010/7/7b/DTU_BB_AntiT2.png" width="570px"  align="center"> </img></p>
 +
<font size="1.5">
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2010/8/84/DTU_BB_AntiT3_graph.png" width="570px"  align="center"> </img></p>
 +
<p align="justify"><b>Figure 7</b>: The graphs illustrated are a simulation of the expected results from <b>Construct 2</b> and <b>Construct 3</b>. The expected results from <b>Construct 2</b> would be a baseline expression of GFP, as the promoter would continually be repressed. With <b>Construct 3</b>, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.</p>
</td>
</td>
<td width="163px" height="100%" valign="top">
<td width="163px" height="100%" valign="top">

Latest revision as of 17:27, 20 October 2010

Welcome to the DTU iGEM wiki!

Home The Team The Project Parts submitted Modelling Notebook Blog

Introduction

In order to optimize the lab work, we split up the work so that we could have two lab teams working in parallel to design different parts of the switch. We split up the lab work so we had:

  • Team 1: The Repressor - Anti-Repressor Team
  • Team 2: The Terminator - Anti-Terminator Team
The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:

The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:

Repressor Group

The repressor group will be assembling the constructs step-by-step:

Step 1

The construction of a plasmid containing the divergent promoters is the first step, the effect of this will be the uninhibited expression of GFP as illustrated by the green colonies observed in Figure 4.

Figure 3: The initial plasmid constructed is illustrated. The divergent promoters have been inserted into a plasmid and transformed into the electro-competent E.coli cells.

Figure 3: The success of the plasmid construction and transformation is illustrated by the fluorescent green colonies seen on the LB-agar plates.

Step 2

Figure 4: The construction of a plasmid containing the Repressor protein (GogR or GtgR) expressed from the pRM promoter is shown. The continually expressed repressor protein will then inhibit the pR promoter and no GFP will be expressed.

Step 3

Figure 5: The independent plasmid is constructed is shown. This plasmid contains the gene encoding the anti-repressor is found downstream of the promoter induced by arabinose, pBAD.

Results Simulation

Figure 7: The graphs illustrated are a simulation of the expected results from Construct 2 and Construct 3. The expected results from Construct 2 would be a baseline expression of GFP, as the promoter would continually be repressed. With Construct 3, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.

Figure 7: The graphs illustrated are a simulation of the expected results from Construct 2 and Construct 3. The expected results from Construct 2 would be a baseline expression of GFP, as the promoter would continually be repressed. With Construct 3, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.

Retrieved from "http://2010.igem.org/Team:DTU-Denmark/BBrick_Characterisation"