Team:DTU-Denmark/BBrick Characterisation

From 2010.igem.org

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<title>Welcome to the DTU iGEM wiki!</title>
<title>Welcome to the DTU iGEM wiki!</title>
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<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/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/SPL">Synthetic Promoter Library</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Team1">Anti-Repressor group</a></li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/BBrick_Characterisation">Results</a></li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Team2">Anti-Terminator group</a></li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/BBrick_Characterisation">Characterisation of Biobricks</a></li><br>
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<h1>Introduction</h1>
<h1>Introduction</h1>
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<p align="justify">In order to optimise 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>
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<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>
<ul>
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<li>Team 1: <b>The Repressor-AntiRepressor Team</b></li>
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<li>Team 1: <b>The Repressor - Anti-Repressor Team</b></li>
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<li>Team 2: <b>The Terminator-AntiTerminator Team</b></li>
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<li>Team 2: <b>The Terminator - Anti-Terminator Team</b></li>
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The Repressor (Repressor-AntiRepressor) Team is responsible for assembling the construct illustrated below in Figure 1:<br>
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The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:<br>
<p align="center"><img scr=""></img></p>
<p align="center"><img scr=""></img></p>
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The AntiTerminator (Terminator-AntiTerminator) Team is responsible for assembling the construct illustrated in Figure 2:<br>
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The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:<br>
<p align="center"><img scr=""></img></p>
<p align="center"><img scr=""></img></p>
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<h3>Step 2</h3>
<h3>Step 2</h3>
<p align="center"><img src="https://static.igem.org/mediawiki/2010/e/ef/DTU_BB_Repressor1.png" width="570px"  align="center"> </img></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2010/e/ef/DTU_BB_Repressor1.png" width="570px"  align="center"> </img></p>
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<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>
<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>
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<h3>Step 3</h3>
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<font size="1.5">
<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="center"><img src="https://static.igem.org/mediawiki/2010/7/7c/DTU_BB_Repressor2.png" width="570px"  align="center"> </img></p>
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<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>
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<h3>Results Simulation</h3>
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<font size="1.5">
<p align="center"><img src="https://static.igem.org/mediawiki/2010/3/34/DTU_BB_Repressor3_graph.png" width="570px"  align="center"> </img></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2010/3/34/DTU_BB_Repressor3_graph.png" width="570px"  align="center"> </img></p>
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<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>
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</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/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>
<p align="center"><img src="https://static.igem.org/mediawiki/2010/7/7b/DTU_BB_AntiT2.png" width="570px"  align="center"> </img></p>
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<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="center"><img src="https://static.igem.org/mediawiki/2010/8/84/DTU_BB_AntiT3_graph.png" width="570px"  align="center"> </img></p>
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<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>
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Latest revision as of 17:27, 20 October 2010

Welcome to the DTU iGEM wiki!


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.