Team:DTU-Denmark/Results

From 2010.igem.org

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<ul>
<ul>
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<font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Construction" target="_blank">Construction of BioBricks</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Construction" >Construction of BioBricks</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization" target="_blank">Characterization</a>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization" >Characterization</a>
<ul>
<ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization_Strategy" target="_blank">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization_Strategy" >Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization_Results" target="_blank">Results</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section#Characterization_Results" >Results</a></li>
</ul>
</ul>
</li>
</li>
</ul><br></font>
</ul><br></font>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section">Anti-Terminator Section</a></li>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section">Terminator - Anti-Terminator Section</a></li>
<ul>
<ul>
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<font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Construction" target="_blank">Construction of BioBricks</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization" >Characterization</a>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization" target="_blank">Characterization Results</a>
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<ul>
<ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization_Strategy" target="_blank">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization_Strategy" >Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization_Results" target="_blank">Results</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Characterization_Results" >Results</a></li>
</ul>
</ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#biobricks" >Construction of BioBricks</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section#Construction" >Construction of Parts</a></li>
</li>
</li>
</ul><br></font>
</ul><br></font>
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<ul>
<ul>
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<font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Construction" target="_blank">Construction of BioBricks</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Construction" >Construction of BioBricks</a>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization" target="_blank">Characterization Results</a>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization" >Characterization Results</a>
<ul>
<ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization_Strategy" target="_blank">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization_Strategy" >Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization_Results" target="_blank">Results</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section#Characterization_Results" >Results</a></li>
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</ul>
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<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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  <font color="#990000" face="arial" size="5">
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<br>  
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<b>Introduction</b><br><br>
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  </font>
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 +
 
 +
 
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<p align="justify">The key focus in this project was put on the characterization of the regulatory elements that our switch comprises of; repressor and anti-repressor from the Gifsy1 and Gifsy2 phages and terminator and anti-terminator from lambda and P22 phages. </p>
 +
 
 +
<p align="justify">As a proof of concept for the regulatory systems several constructs were made in low-copy-number plasmids. By means of the reporter proteins GFP and RFP we were able to investigate our colonies under a microscope and in a fluorometer to see if the expected reporters were expressed and thereby if the BioBricks work as expected. In addition to that, we also ran some measurements in a BioLector which can detect both OD and fluorescence simultaneously and thus also makes it probable that our BioBricks work as expected.</p>
 +
 
 +
<p align="justify">The measurements carried out in the BioLector also form the basis of the characterization of the BioBricks. From the plots derived from the measurements we are able to see the effect of the Gifsy1 and Gifsy2 promoters when the repressor is expressed.</p>
 +
 
 +
<p align="justify">As a means of characterizing the anti-terminator N we developed a Synthetic Promoter Library (SPL) which is an ideal way of fine-tuning gene expression. The SPL comprises of a wide variety of promoters with different promoter strengths and by cloning the SPL in front of the N protein it is possible to determine the right level of expression and thus the right promoter. However, as the idea for the SPL was evolved it became clear that the SPL could indeed be used to characterize and/or fine-tune other BioBricks and thus the idea of making the SPL as a standard was developed.</p>
 +
 
 +
<p align="justify">The experimental work has been divided into three parts. Here you can find results and characterization of:</p>
 +
 
<ul>
<ul>
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<li>Team 1: <b>The Repressor - Anti-Repressor Section</b></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section ">Repressors and anti-repressors </a></li>
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<li>Team 2: <b>The Terminator - Anti-Terminator Section</b></li>
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<li><a href=" https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section">Terminators and anti-terminators </a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section ">Synthetic Promoter Library </a></li>
</ul>
</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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<font size="1.5">
 
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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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<font size="1.5">
 
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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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<font size="1.5">
 
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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>
 
-
</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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<font size="1.5">
 
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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>
 
-
<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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</td>
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<td width="163px" height="100%" valign="top">

Latest revision as of 01:20, 28 October 2010

Welcome to the DTU iGEM wiki!



Introduction

The key focus in this project was put on the characterization of the regulatory elements that our switch comprises of; repressor and anti-repressor from the Gifsy1 and Gifsy2 phages and terminator and anti-terminator from lambda and P22 phages.

As a proof of concept for the regulatory systems several constructs were made in low-copy-number plasmids. By means of the reporter proteins GFP and RFP we were able to investigate our colonies under a microscope and in a fluorometer to see if the expected reporters were expressed and thereby if the BioBricks work as expected. In addition to that, we also ran some measurements in a BioLector which can detect both OD and fluorescence simultaneously and thus also makes it probable that our BioBricks work as expected.

The measurements carried out in the BioLector also form the basis of the characterization of the BioBricks. From the plots derived from the measurements we are able to see the effect of the Gifsy1 and Gifsy2 promoters when the repressor is expressed.

As a means of characterizing the anti-terminator N we developed a Synthetic Promoter Library (SPL) which is an ideal way of fine-tuning gene expression. The SPL comprises of a wide variety of promoters with different promoter strengths and by cloning the SPL in front of the N protein it is possible to determine the right level of expression and thus the right promoter. However, as the idea for the SPL was evolved it became clear that the SPL could indeed be used to characterize and/or fine-tune other BioBricks and thus the idea of making the SPL as a standard was developed.

The experimental work has been divided into three parts. Here you can find results and characterization of: