Team:DTU-Denmark/Results

From 2010.igem.org

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<h1>Introduction</h1>
<h1>Introduction</h1>
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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>
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<p align="justify">As previously described in the design of the <a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Engineering">switch</a>, each half switch contains a nut site followed by a terminator, as well as an anti-terminator. The roles of these parts are to increase the stability of the current state and thereby also the robustness of the switch. As the PRM promoter is not very well repressed by the GogR/GtgR repressors, transcription is promoted even in their presence. If transcription was allowed to continue to the anti-repressor located on the inactive half of the switch, the switch could change state spontaneously. The terminator is placed in front of the anti-repressor to ensure that this does not happen. The anti-terminator of the active state is expressed, allowing continued transcription past the terminator. </p>
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<ul>
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<li>Team 1: <b>The Repressor - Anti-Repressor Section</b></li>
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<li>Team 2: <b>The Terminator - Anti-Terminator Section</b></li>
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</ul>
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The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:<br>
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<p align="center"><img scr=""></img></p>
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The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:<br>
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<p align="center"><img scr=""></img></p>
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</p>
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<h1>Repressor Group</h1>
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<p align="justify">The repressor group will be assembling the constructs step-by-step:</p>
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<h3>Step 1</h3>
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<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>
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<p align="center"><img scr=""></img></p>
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<font size="1.5">
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<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>
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<p align="center"><img scr=""></img></p>
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<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>
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</font>
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<h3>Step 2</h3>
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<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>
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</font>
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<h3>Step 3</h3>
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<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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</font>
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<h3>Results Simulation</h3>
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<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>
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/a/a0/DTU_BB_AntiT1.png" width="570px"  align="center"> </img></p>
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<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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<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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Revision as of 14:22, 25 October 2010

Welcome to the DTU iGEM wiki!


Introduction

As previously described in the design of the switch, each half switch contains a nut site followed by a terminator, as well as an anti-terminator. The roles of these parts are to increase the stability of the current state and thereby also the robustness of the switch. As the PRM promoter is not very well repressed by the GogR/GtgR repressors, transcription is promoted even in their presence. If transcription was allowed to continue to the anti-repressor located on the inactive half of the switch, the switch could change state spontaneously. The terminator is placed in front of the anti-repressor to ensure that this does not happen. The anti-terminator of the active state is expressed, allowing continued transcription past the terminator.