Team:DTU Denmark/introduction private securkey Dhjg1mab2ak47

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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark" >Home</a></font> </td>
    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/team" >The Team</a> </font></td>
    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/project" >The Project</a> </font></td>
    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/parts" >Parts submitted</a> </font></td>
    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/modelling">Modelling</a></font> </td>
    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/notebook" title="Day to day lab activity">Notebook</a> 

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   The redoxilator<br><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/theory" CLASS=leftbar>- Introduction</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/yeast" CLASS=leftbar>- Results</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/practicalapproach" CLASS=leftbar>- Applications and perspectives</a><br><br>
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    <br>The USER<sup>TM</sup> assembly standard<br><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprinciple" CLASS=leftbar>- Principle</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/USERconcept" CLASS=leftbar>- Proof of concept</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/USERmanual" CLASS=leftbar>- Manual</a><br><br>
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    <br>USER<sup>TM</sup> fusion primer design software<br><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogram" CLASS=leftbar>- Abstract</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprograminstructions" CLASS=leftbar>- Instructions</a><br>
    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogramoutputformat" CLASS=leftbar>- Output format</a><br>
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   <b>The project</b><br><br><br>
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<font size="4"><b>Genetic design</b></font><br><br>

<p>We have successfully designed and physically constructed a genetic system that couples the intracellular NAD+/NADH level to the gene expression of a reporter protein. The system has potentially many applications including in vivo online monitoring of the redox poise, production optimization and cancer research with yeast as a model organism (see <i>Applications</i>).</p>
<p>The NAD+/NADH ratio is sensed by a system originating in <i>Streptomyces coellicolor</i>. In <i>S. coellicolor </i>the protein REX is a repressor and controls the gene expression of multiple genes by recognizing and binding to a specific DNA-sequence termed ROP (<u>R</u>ex <u>op</u>erator). NAD+ and NADH compete to associate with Rex, but only a REX:NAD+ association can bind the ROP DNA-sequence (Brekasis and Paget, 2003).</p>
<p>In <i>S. coellicolor</i> REX DNA binding represses expression of target genes, by physically hindering RNA-polymerases from binding the promoter. As the transcription machinery of eukaryotes is different and more complicated, there are no guarantee that repression will be effective in eukaryotes. REX has therefore been fused to a eukaryotic transcriptional activator, a widely used technique applied for the investigation of the GAL proteins and other systems (Sadowski et al. 1988). The REX-activator fusion-protein is able to bind the ROB sequence placed upstream of a minimal eukaryotic promoter that only supports transcription upon activation. A certain NAD+/NADH ratio will activate the Redoxilator to recognize the ROB promoter, resulting in transcription of the reporter gene.</p>

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[[Image:genedesignA.jpg.redox.jpg|400px|thumb|center|The redox coupled system]]
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[[Image:genedesignB.jpg.redox.jpg|400px|thumb|center|The redox coupled system]]
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<p align="justify"><i><b>Gene design and redox regulation</b><br>
<b>A:</b> The Rex gene has been fused to an activator domain and is transcribed constitutively, leading to constant concentration of the Rex-activator protein in the cell. The ROB sequence and a minimal promoter is followed by a reporter gene, which is only be transcribed when the REX-activator fusion protein is bound to the promoter. <b>B:</b> The REX-activator only binds the ROB DNA sequence under the condition of having NAD+ bound. Under these circumstances the fused activator domain summons the RNA polymerase and the reporter gene is transcribed</i></p><br>


<p><b>Design specifications</b><b> </b><br />
The genetic system consists of two synthetic genes: one coding for the REX-activator fusion protein, and one coding for a yeast optimized GFP gene under control of ROB fused to a minimal promoter. With this system GFP is only be expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.<b></b></p>

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[[Image:dnalevel_construct.png|500px|thumb|center|<i><b>Schematic representation of the synthetic genetic system on the DNA-level</b></i>]]

[[Image:redoxilator_biobrick_style.png|500px|thumb|center|<i><b>Schematic representation of the synthetic genetic system on the DNA-level</b></i>]]


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<p>The genetic elements and the requirements they need to fulfill are listed in the following table. The detailed description of the used genetic elements will not be made publicly available due to IP rights.</p>
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<p>Description and requirements of genetic elements.</p>

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	      <td width="141" valign="top"><p><b>Genetic element</b></td>
	      <td width="295" valign="top"><p><b>Required function</b></p></td>
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	      <td width="141" valign="top"><p>Constitutive promoter</p></td>
	      <td width="295" valign="top"><p>Constitutive expression of   REX-Activator fusion protein</p></td>
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	      <td width="141" valign="top"><p>Kozak sequence</p></td>
	      <td width="295" valign="top"><p>Ribosome start-codon   recognition and enhanced initiating of translation</p></td>
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	      <td width="141" valign="top"><p>REX</p></td>
	      <td width="295" valign="top"><p>REX (<u>re</u>do<u>x</u> regulator) that binds to   ROP at high NAD+/NADH ratio </p></td>
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	      <td width="141" valign="top"><p>Linker</p></td>
	      <td width="295" valign="top"><p>To couple two protein   domains without disrupting their individual functions</p></td>
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	      <td width="141" valign="top"><p>Activator domain</p></td>
	      <td width="295" valign="top"><p>Protein domain able to   activate transcription in eukaryotes in proximity of a minimal promoter.</p></td>
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	      <td width="141" valign="top"><p>Nuclear Localization   Sequence (NLS)</p></td>
	      <td width="295" valign="top"><p>Translocation of the   REX-Activator protein to the nucleus</p></td>
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	      <td width="141" valign="top"><p>Terminator 1</p></td>
	      <td width="295" valign="top"><p>Sequence that terminates   transcription</p></td>
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	      <td width="141" valign="top"><p>&nbsp;</p></td>
	      <td width="295" valign="top"><p>&nbsp;</p></td>
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	      <td width="141" valign="top"><p>ROB</p></td>
	      <td width="295" valign="top"><p>DNA sequence that REX binds   at high NAD+/NADH ratio</p></td>
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	      <td width="141" valign="top"><p>Minimal promoter</p></td>
	      <td width="295" valign="top"><p>Promoter devoid of   regulatory motifs. Only expression if an activator is bound upstream.</p></td>
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	      <td width="141" valign="top"><p>GFP</p></td>
	      <td width="295" valign="top"><p>The amount of green   fluorescent protein can be quantitatively measured.</p></td>
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	      <td width="141" valign="top"><p>Degradation signal</p></td>
	      <td width="295" valign="top"><p>Ensures fast degradation of   GFP</p></td>
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	      <td width="141" valign="top"><p>Terminator 2</p></td>
	      <td width="295" valign="top"><p>Terminates transcription.   Different from terminator 1 to avoid direct repeats, which can cause the DNA to loop out.</p></td>
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	 <b>Synthetic Biology</b><br><br>


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	<p align="left"><i>“Synthetic Biology is an art of engineering new biological systems that don’t exist in nature.”</i><br></p>

	<p align="right"><i>-Paras Chopra & Akhil Kamma</i><br><br></p>

	<p>In nature, biological molecules work together in complex systems to serve purposes of the cell. In synthetic biology these molecules are used as individual functional units that are combined to form tailored systems exhibiting complex dynamical behaviour. From ‘design specifications’ generated from computational modelling, engineering-based approaches enables the construction of such new specified gene-regulatory networks. The ultimate goal of synthetic biology is to construct systems that gain new functions, and the perspectives of the technology are enormous. It has already been used in several medical projects2 and is predicted to play a major role in biotech-production and environmental aspects.</p>

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 Comments or questions to the team? Please <a href="mailto:igem@bio.dtu.dk" CLASS=email>Email us</a> -- Comments of questions to webmaster? Please <a href="mailto:lronn@bio.dtu.dk" CLASS=email>Email us</a>
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