http://2007.igem.org/wiki/index.php?title=Special:Contributions/RGaber&feed=atom&limit=50&target=RGaber&year=&month=2007.igem.org - User contributions [en]2024-03-28T09:48:09ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/Ljubljana/GabrielaPanterLjubljana/GabrielaPanter2007-11-23T19:02:51Z<p>RGaber: </p>
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<h3><span>Gabriela Panter</span></h3><br />
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PhD student, National Institute of Chemistry Slovenia, Department of Biotechnology<br />
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Contact: gabriela.panter@ki.si<br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/MatejaMancekKeberLjubljana/MatejaMancekKeber2007-11-23T19:02:29Z<p>RGaber: </p>
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<link rel="stylesheet" href="/igem07/index.php?title=User:RGaber/stylesheet/stylenew_zadaj.css&action=raw&ctype=text/css" type="text/css" /><br />
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<h3><span>Mateja Manček Keber</span></h3><br />
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Postdoctoral researcher, National Institute of Chemistry Slovenia, Department of Biotechnology<br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
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<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3><br />
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<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
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<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li><br />
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<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
<center><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/KarolinaIvicakLjubljana/KarolinaIvicak2007-11-23T19:02:08Z<p>RGaber: </p>
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<title>Company Name</title><br />
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<link rel="stylesheet" href="/igem07/index.php?title=User:RGaber/stylesheet/stylenew_zadaj.css&action=raw&ctype=text/css" type="text/css" /><br />
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<h3><span>Karolina Ivičak</span></h3><br />
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<img src="https://static.igem.org/mediawiki/2007/f/f1/Karolina.jpg" width="600" height="450"><br />
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PhD student, National Institute of Chemistry Slovenia, Department of Biotechnology<br />
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Contact: karolina.ivicak@ki.si<br />
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|<a href="https://2007.igem.org/Ljubljana">Home</a>|<br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
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<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
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<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li><br />
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</ul><br />
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<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<ul><br />
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<br><br><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
<center><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/MojcaBencinaLjubljana/MojcaBencina2007-11-23T19:01:45Z<p>RGaber: </p>
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<title>Company Name</title><br />
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<link rel="stylesheet" href="/igem07/index.php?title=User:RGaber/stylesheet/stylenew_zadaj.css&action=raw&ctype=text/css" type="text/css" /><br />
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<h3><span>Mojca Benčina</span></h3><br />
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<img src="https://static.igem.org/mediawiki/2007/2/25/Mojca.jpg"<br />
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Molecular biologist with great passion for sports. Crazy scientist running from one to another problem. <br><br><br />
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Researcher at Department of Biotechnology, National Institute of Chemistry, Slovenia.<br><br><br />
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Contact: mojca.bencina@ki.si <br />
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|<a href="https://2007.igem.org/Ljubljana">Home</a>|<br />
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<div id="lmenu"><br />
<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
<br />
<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li><br />
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<br />
</ul><br />
</div><br />
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<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<ul><br />
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<br><br><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
<center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/Ljubljana" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/Ljubljana" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" /><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/MarkoDolinarLjubljana/MarkoDolinar2007-11-23T19:01:20Z<p>RGaber: </p>
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<h3><span>Marko Dolinar</span></h3><br />
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<br><img src="https://static.igem.org/mediawiki/2007/6/60/Moi1.jpg"><br><br><br />
Together with <a href="https://2007.igem.org/Ljubljana/RomanJerala">Roman Jerala</a>, I am co-supervisor of the Slovenian team. Five out of seven undergraduates in our team are or were my students. I teach molecular biology within the Biochemistry course and Recombinant DNA Technology theoretical and practical course here at the <a href="http://www.uni-lj.si/en/">University of Ljubljana</a> <a href="http://www.fkkt.uni-lj.si/en/">Faculty of Chemistry and Chemical Technology</a>. Our Biochemistry Chair presents itself <a href="http://openwetware.org/wiki/FCCT_Biochemistry_Lab">on OWW</a>.<br><br />
If you are fluent in Slovenian, visit my <a href="http://novebiologije.blogspot.com/">blog "New Biologies"</a> where I present and comment news from the field of molecular, synthetic, systems and related biologies.<br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/RomanJeralaLjubljana/RomanJerala2007-11-23T19:00:56Z<p>RGaber: </p>
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<h3><span>Roman Jerala</span></h3><br />
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Our department can't fit into this small boat anymore.<br />
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prof. Roman Jerala PhD<br><br />
head<br><br />
Department of biotechnology<br><br />
National Institute of Chemistry<br><br />
Hajdrihova 19<br><br />
POB 660<br><br />
1000 Ljubljana<br><br />
SLOVENIA<br><br />
tel. +386 1 476 0335<br><br />
fax. +386 1 476 0300<br><br />
Contact: roman jerala at nic si<br><br />
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<a href="http://www.ki.si/index.php?id=218&L=1">Lab at NIC</a><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/AndrejOndrackaLjubljana/AndrejOndracka2007-11-23T19:00:28Z<p>RGaber: </p>
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<h3><span>Andrej Ondračka</span></h3><br />
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At the beginning of my studies I became interested in structural biology and biomolecular spectroscopy. Later, I also discovered the magical world of synthetic and systems biology. Currently, I am equally interested in both.<br><br />
Besides science, I like music, philosophy, photography, and my great passion, travelling.<br><br />
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For any information about me don't hesitate to contact me on andrej_ondracka (at) yahoo.com<br />
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<h3><span>Anja Korenčič</span></h3><br />
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<td align="justify">Music is one of the most important aspects of my life apart from biochemistry; I play clarinet in Vevce Wind Orchestra and percussion in newly founded percussion group RitemDaBit. If you are interested, you can check my orchestra's <a href="http://www.orkester.si">home page</a> with extensive photogallery. I also love travelling and photography.<br><br><br />
Contact: anja_korencic(at)yahoo.com<br />
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<h3><span>Katja Kolar</span></h3><br />
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<td> <img src ="https://static.igem.org/mediawiki/2007/d/d8/Katja.jpg"></td><br />
<td align="justify"> Most probably a future scientist in biochemistry (somewhere with lots of links with medicine or physics), if not, I'll live in a wonderful world of architecture and math. <br />
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Through the work for iGEM I nearly became a specialist for preparing cells for microscopy, but besides lab I also like painting, philosophy, theater, music (flute) and backpacking, which is a must.<br />
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More questions about me? Feel free to write: katja.kolar (at) gmail.com </td><br />
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<h3><span>Saša Jereb</span></h3><br />
<p class="p1"><span><br />
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<img src="https://static.igem.org/mediawiki/2007/2/2c/Sasa.jpg"><br />
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<br><br>Undergraduate student of Biochemistry.<br><br />
I decided to join iGEM team because I wanted to know what the term »synthetic biology« means in the lab. I enjoyed all the work a lot, except making agarose gels. In the future I'll work in a lab where they are buying them.<br><br />
When I grow up I'll be working on improving human health, in case I'll become a biochemist.<br />
In case not, I'll be an illustrator.<br />
If you want to know more about our project mail me: sasajereb at gmail. <br />
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<h3><span>Rok Gaber</span></h3><br />
<p class="p1"><span><br />
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<img src="https://static.igem.org/mediawiki/2007/e/ea/RokGaberSEPIA.jpg" width="633" height="474"><br><br />
<b>Since a droplet of Ethidium Bromide gushed into my eyes and I tried to deactivate it with UV lamp, I have to wear sunglasses all the time.</b><br><br><br />
Last year of microbiology studies at Biotechnical Faculty (University in Ljubljana). Besides synthetic biology I am also interested in alternative fuels, microbic expression systems and other areas of industrial biotechnology.<br><br><br />
<b>Contact:</b><br><br />
rok.gaber(at)gmail.com <br />
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<h3><span>Peter Cimermančič</span></h3><br />
<p class="p1"><span><br />
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<img src="https://static.igem.org/mediawiki/2007/5/5d/Peter4.jpg"<br />
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Computational and system biology is what I like to do. I just cannot wait to results of wet lab experiments (<i>in silico</i> work is much faster). Team in wet lab is also a kind of system with which I had a lot of fun. So please give me both. <br />
<br><br><br />
Contact: peter.cimermancic@gmail.com<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<h3><span>Marko Bitenc</span></h3><br />
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<td>I am in my final year of study at University of Ljubljana and in parallel I am also an MSc student in cellular and molecular biotechnology at Wageningen University.<br />
One special thing about me is that I never stop looking for new challenges. I could probably write all day about my interests, but then you would not read this.<br />
Anyways, temporarily I am trying to combine science and economy and make some added value out of it. <br><br><br />
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marko@febodesigns.org<br />
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<b>AIDS - </b> <small>Acquired immune deficiency syndrome is a collection of symptoms and infections resulting from the specific damage to the immune system caused by the human immunodeficiency virus (HIV) in humans. The late stage of the condition leaves individuals susceptible to opportunistic infections and tumors.</small> <br> <br><br />
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<b>Apoptosis - </b> <small> is a form of programmed cell death in multicellular organisms. It is one of the main types of programmed cell death and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death. </small><br><br><br />
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<b>Capsid - </b> <small>is the protective shell of protein that surrounds the genetic material of a virus.<br />
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<b>CCR5 - </b> <small>short for chemokine (C-C motif) receptor 5. CCR5 is predominantly expressed on T cells, macrophages, dendritic cells and microglia. HIV uses CCR5 or another protein, CXCR4, as a co-receptor to enter its target cells. CCR5 is likely the most physiologically important coreceptor during natural infection. </small><br><br><br />
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<b>CD4 - </b> <small>CD4 (cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells. Like many cell surface receptors/markers, CD4 is a member of the immunoglobulin superfamily. CD4 is also a primary receptor used by HIV-1 to gain entry into host T cells.</small><br><br><br />
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<b>CXCR4 - </b> <small>also called fusin, this is one of two co receptors for HIV binding to the human cell, it is embedded in the T Cell membrane. The other co receptor is CCR5. </small><br><br><br />
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<b><i>Env - </i></b> <small>The envelope gene encodes the surface glycoprotein (SU) - transmembrane (TM) polyprotein. <i>Env</i> gene is located in the HIV genome.</small><br><br><br />
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<b><i>Gag - </i></b> <small>The <i>gag</i> (group specific antigen) gene encodes the viral matrix (MA) capsid (CA) and nucleoproteins (NC). <i>Gag</i> gene is located in the HIV genome.</small><br><br><br />
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<b>GFP - </b> <small>protein, comprised of 238 amino acids (26,9 kDa), originally isolated from the jellyfish. When GFP is exposed to blue light it fluoresces green and is therefore easy to detect on confocal microscope.</small><br><br><br />
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<b>gp41 - </b> <small>is a glycoprotein non-covalently bound to gp120, and provides the second step by which HIV enters the cell. It is originally buried within the viral envelope, but when gp120 binds to a CD4 receptor, gp120 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell.</small><br><br><br />
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<b>gp120 - </b> <small>is a glycoprotein exposed on the surface of the HIV envelope. The glycoprotein gp120 is anchored to the viral membrane through non-covalent bonds along with gp41, both coming from a cleaved protein, gp160. It infects any target cell with a CD4 receptor, particularly the helper T-cell, by binding to that receptor.</small><br><br><br />
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<b>HAART - </b> <small>Highly Active AntiRetroviral Therapy. Treatment for human immunodeficiency virus (HIV) infection that uses a combination of several antiretroviral drugs. The drugs inhibit the ability of the virus to multiply in the body, and they slow down the development of AIDS.</small><br><br><br />
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<b>HIV - </b> <small>Human immunodeficiency virus is a retrovirus that can lead to acquired immunodeficiency syndrome (AIDS). HIV infection leads to a progressive reduction in the number of T cells possessing CD4 receptors.</small><br><br><br />
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<b>HIV protease - </b> <small>aspartyl protease that is essential for the life-cycle of HIV. Inhibition of this protease prevents maturation of HIV particles. As such, many drugs have been developed (protease inhibitors) that target this enzyme. </small><br><br><br />
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<b>Integrase - </b> <small>an enzyme produced by a retrovirus (including HIV) that enables its genetic material to be integrated into the DNA of the infected cell. It is also produced by viruses containing double stranded DNAs for the same purpose. </small><br><br><br />
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<b>LTR - </b> <small>long terminal repeats (LTRs) are found in retroviral DNA flanking functional genes. The LTRs are partially transcribed into an RNA intermediate, followed by reverse transcription into complementary DNA (cDNA) and ultimately dsDNA (double-stranded DNA) with full LTRs. The LTRs then mediate integration of the retroviral DNA via an LTR specific transposase called integrase into another region of the chromosome. This is the basic mechanism used by HIV. LTRs also act as promoters/enhancers - when integrated into the host genome they influence the cell machinery which transcribes DNA to alter the amount of transcription which occurs</small><br><br><br />
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<b>NRTI - </b> <small>analogs of nucleosides/nucleotides (building blocks necessary for DNA synthesis). They inhibit the reverse transcriptase of HIV, thus inhibiting its replication and leading to decreased viral loads.</small><br><br><br />
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<b>NNRTI - </b> <small>non-nucleoside reverse transcriptase inhibitors, a family of reverse transcriptase inhibitors used as anti AIDS treatment.<br />
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<b>Polyprotein - </b> <small>protein that is cleaved after translation to produce several functionally distinct proteins. </small><br><br><br />
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<b><i>Pol - </i></b> <small>encodes the reverse transcriptase (RT) and an integrase (IN). <i>Pol</i> gene is located in the HIV genome.</small><br><br><br />
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<b>Retrovirus - </b> <small>is any virus belonging to the viral family Retroviridae. They are enveloped viruses possessing a RNA genome and replicate via DNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA, which can then be integrated into the host's genome with an integrase enzyme. The virus then replicates as part of the cell's DNA.</small><br><br><br />
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<b>Split protein system - </b> <small>Experimental procedure to detect protein-protein interactions that uses two fusion proteins (“hybrids”) which reconstitute an active transcription factor (or some other active protein) when the two fusion proteins interact. </small><br><br><br />
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<b>T-cells - </b> <small>belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They attack virus-infected cells, foreign cells and cancer cells. T-cells also produce a number of substances that regulate the immune response.</small><br><br><br />
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<b>T7 RNA polymerase - </b> <small>catalyzes the formation of RNA in the 5'→ 3' direction. T7 RNA polymerase is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter.</small><br><br><br />
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<b>TEV protease - </b> <small>highly site-specific protease that is found in the Tobacco Etch Virus (TEV). It is a cysteine protease. Advantages of this enzyme are its high specificity and its high activity rate.</small><br><br><br />
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<b>Ubiquitin - </b> <small> highly conserved small regulatory protein that is ubiquitous in eukaryotes. Ubiquitination refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The most prominent function of ubiquitin is labeling proteins for proteasomal degradation.</small><br><br><br><br />
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<h3><span>References</span></h3><br />
<p class="p1"><span><br />
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Drugs Used in the Treatment of HIV Infection. 2007. FDA.<br><br />
http://www.fda.gov/oashi/aids/virals.html (October 2007)<br><br><br />
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Heckman K.L., Pease L.P. 2007. Gene splicing and mutagenesis by PCR-driven overlap<br />
Extension. <br> Nature Protocols, 2, 4: 924-932<br><br><br />
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Johnsson N., Varshavsky A. 1994. Split ubiquitin as a sensor of protein interactions in vivo.<br> Proceedings of the National Academy of Sciences, 91: 10340–10344<br><br><br />
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Lieber A., Kiessling U., Strauss M. 1989. High level gene expression in mammalian cells by a nuclear T7-phage RNA polymerase. <br> Nucleic Acids Research, 17, 21: 8485-8493 <br><br><br />
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Stagljar I., Fields S. 2002. Analysis of membrane protein interactions using yeast-based technologies.<br> TRENDS in Biochemical Sciences, 27, 11: 559-563<br><br><br />
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Vocero-Akbani A.M., Heyden N.V., Lissy N.A., Ratner L., Dowdy S.F. 1999. Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. <br> Nature Medicine, 5, 1: 29-33<br><br><br />
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Wehr M.C., Laage R., Bolz U., Fischer T.M., Grunewald S., Scheek S., Bach A., Nave K.A., Rossner M.J. 2006. Monitoring regulated protein-protein interactions using split TEV. <br> Nature Methods, 3, 12: 985-993 <br><br><br />
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<td><a href="http://www.lek.si/eng/"> Lek - a Sandoz Company</a></td><br />
<td><a href="http://www.lek.si/eng/"><img border="0" src="https://static.igem.org/mediawiki/2007/9/9a/Lek-logo.jpg" width="200" height="75"> <br></a><br></td><br />
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<td><a href="http://www.krka.si/en/"> Krka Pharmaceutical Company </a></td><br />
<td><a href="http://www.krka.si/en/"><img border="0" src="https://static.igem.org/mediawiki/2007/0/0e/Krka-logo.gif"> <br></a><br></td><br />
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<td><a href="http://www.geneart.com/"> GeneArt </a></td><br />
<td><a href="http://www.geneart.com/"><img border="0" src="https://static.igem.org/mediawiki/2007/d/d6/GeneArt.jpg" width="193" height="42"><br></a><br></td><br />
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<td><a href="http://www.mvzt.gov.si/en/"><br />
Ministry of Higher Education,Science and Technology of Slovenia</a></td><br />
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<h3><span>Glossary</span></h3><br />
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<b>AIDS - </b> <small>Acquired immune deficiency syndrome is a collection of symptoms and infections resulting from the specific damage to the immune system caused by the human immunodeficiency virus (HIV) in humans. The late stage of the condition leaves individuals susceptible to opportunistic infections and tumors.</small> <br> <br><br />
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<b>Apoptosis - </b> <small> is a form of programmed cell death in multicellular organisms. It is one of the main types of programmed cell death and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death. </small><br><br><br />
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<b>Capsid - </b> <small>is the protective shell of protein that surrounds the genetic material of a virus.<br />
</small><br><br><br />
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<b>CCR5 - </b> <small>short for chemokine (C-C motif) receptor 5. CCR5 is predominantly expressed on T cells, macrophages, dendritic cells and microglia. HIV uses CCR5 or another protein, CXCR4, as a co-receptor to enter its target cells. CCR5 is likely the most physiologically important coreceptor during natural infection. </small><br><br><br />
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<b>CD4 - </b> <small>CD4 (cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells. Like many cell surface receptors/markers, CD4 is a member of the immunoglobulin superfamily. CD4 is also a primary receptor used by HIV-1 to gain entry into host T cells.</small><br><br><br />
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<b>CXCR4 - </b> <small>also called fusin, this is one of two co receptors for HIV binding to the human cell, it is embedded in the T Cell membrane. The other co receptor is CCR5. </small><br><br><br />
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<b><i>Env - </i></b> <small>The envelope gene encodes the surface glycoprotein (SU) - transmembrane (TM) polyprotein. <i>Env</i> gene is located in the HIV genome.</small><br><br><br />
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<b><i>Gag - </i></b> <small>The <i>gag</i> (group specific antigen) gene encodes the viral matrix (MA) capsid (CA) and nucleoproteins (NC). <i>Gag</i> gene is located in the HIV genome.</small><br><br><br />
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<b>GFP - </b> <small>protein, comprised of 238 amino acids (26,9 kDa), originally isolated from the jellyfish. When GFP is exposed to blue light it fluoresces green and is therefore easy to detect on confocal microscope.</small><br><br><br />
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<b>gp41 - </b> <small>is a glycoprotein non-covalently bound to gp120, and provides the second step by which HIV enters the cell. It is originally buried within the viral envelope, but when gp120 binds to a CD4 receptor, gp120 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell.</small><br><br><br />
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<b>gp120 - </b> <small>is a glycoprotein exposed on the surface of the HIV envelope. The glycoprotein gp120 is anchored to the viral membrane through non-covalent bonds along with gp41, both coming from a cleaved protein, gp160. It infects any target cell with a CD4 receptor, particularly the helper T-cell, by binding to that receptor.</small><br><br><br />
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<b>HAART - </b> <small>Highly Active AntiRetroviral Therapy. Treatment for human immunodeficiency virus (HIV) infection that uses a combination of several antiretroviral drugs. The drugs inhibit the ability of the virus to multiply in the body, and they slow down the development of AIDS.</small><br><br><br />
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<b>HIV - </b> <small>Human immunodeficiency virus is a retrovirus that can lead to acquired immunodeficiency syndrome (AIDS). HIV infection leads to a progressive reduction in the number of T cells possessing CD4 receptors.</small><br><br><br />
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<b>HIV protease - </b> <small>aspartyl protease that is essential for the life-cycle of HIV. Inhibition of this protease prevents maturation of HIV particles. As such, many drugs have been developed (protease inhibitors) that target this enzyme. </small><br><br><br />
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<b>Integrase - </b> <small>an enzyme produced by a retrovirus (including HIV) that enables its genetic material to be integrated into the DNA of the infected cell. It is also produced by viruses containing double stranded DNAs for the same purpose. </small><br><br><br />
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<b>LTR - </b> <small>long terminal repeats (LTRs) are found in retroviral DNA flanking functional genes. The LTRs are partially transcribed into an RNA intermediate, followed by reverse transcription into complementary DNA (cDNA) and ultimately dsDNA (double-stranded DNA) with full LTRs. The LTRs then mediate integration of the retroviral DNA via an LTR specific transposase called integrase into another region of the chromosome. This is the basic mechanism used by HIV. LTRs also act as promoters/enhancers - when integrated into the host genome they influence the cell machinery which transcribes DNA to alter the amount of transcription which occurs</small><br><br><br />
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<b>NRTI - </b> <small>analogs of nucleosides/nucleotides (building blocks necessary for DNA synthesis). They inhibit the reverse transcriptase of HIV, thus inhibiting its replication and leading to decreased viral loads.</small><br><br><br />
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<b>NNRTI - </b> <small>non-nucleoside reverse transcriptase inhibitors, a family of reverse transcriptase inhibitors used as anti AIDS treatment.<br />
</small><br><br><br />
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<b>Polyprotein - </b> <small>protein that is cleaved after translation to produce several functionally distinct proteins. </small><br><br><br />
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<b><i>Pol - </i></b> <small>encodes the reverse transcriptase (RT) and an integrase (IN). <i>Pol</i> gene is located in the HIV genome.</small><br><br><br />
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<b>Retrovirus - </b> <small>is any virus belonging to the viral family Retroviridae. They are enveloped viruses possessing a RNA genome and replicate via DNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA, which can then be integrated into the host's genome with an integrase enzyme. The virus then replicates as part of the cell's DNA.</small><br><br><br />
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<b>Split protein system - </b> <small>Experimental procedure to detect protein-protein interactions that uses two fusion proteins (“hybrids”) which reconstitute an active transcription factor (or some other active protein) when the two fusion proteins interact. </small><br><br><br />
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<b>T-cells - </b> <small>belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They attack virus-infected cells, foreign cells and cancer cells. T-cells also produce a number of substances that regulate the immune response.</small><br><br><br />
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<b>T7 RNA polymerase - </b> <small>catalyzes the formation of RNA in the 5'→ 3' direction. T7 RNA polymerase is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter.</small><br><br><br />
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<b>TEV protease - </b> <small>highly site-specific protease that is found in the Tobacco Etch Virus (TEV). It is a cysteine protease. Advantages of this enzyme are its high specificity and its high activity rate.</small><br><br><br />
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<b>Ubiquitin - </b> <small> highly conserved small regulatory protein that is ubiquitous in eukaryotes. Ubiquitination refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The most prominent function of ubiquitin is labeling proteins for proteasomal degradation.</small><br><br><br><br />
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<h3><span>References</span></h3><br />
<p class="p1"><span><br />
<br />
Drugs Used in the Treatment of HIV Infection. 2007. FDA.<br><br />
http://www.fda.gov/oashi/aids/virals.html (October 2007)<br><br><br />
<br />
Heckman K.L., Pease L.P. 2007. Gene splicing and mutagenesis by PCR-driven overlap<br />
Extension. <br> Nature Protocols, 2, 4: 924-932<br><br><br />
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Johnsson N., Varshavsky A. 1994. Split ubiquitin as a sensor of protein interactions in vivo.<br> Proceedings of the National Academy of Sciences, 91: 10340–10344<br><br><br />
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Lieber A., Kiessling U., Strauss M. 1989. High level gene expression in mammalian cells by a nuclear T7-phage RNA polymerase. <br> Nucleic Acids Research, 17, 21: 8485-8493 <br><br><br />
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Stagljar I., Fields S. 2002. Analysis of membrane protein interactions using yeast-based technologies.<br> TRENDS in Biochemical Sciences, 27, 11: 559-563<br><br><br />
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Vocero-Akbani A.M., Heyden N.V., Lissy N.A., Ratner L., Dowdy S.F. 1999. Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. <br> Nature Medicine, 5, 1: 29-33<br><br><br />
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Wehr M.C., Laage R., Bolz U., Fischer T.M., Grunewald S., Scheek S., Bach A., Nave K.A., Rossner M.J. 2006. Monitoring regulated protein-protein interactions using split TEV. <br> Nature Methods, 3, 12: 985-993 <br><br><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/teamLjubljana/team2007-11-23T18:55:10Z<p>RGaber: </p>
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<h3><span>iGEM 2007 Team Slovenia </span></h3><br />
</span></p><br />
> Click face to find out more about team members.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/7/71/SLOteamOctober_small.jpg" <br />
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usemap="#teammap"><br />
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coords="25,31,119,214" <br />
alt="Roman Jerala"<br />
href="https://2007.igem.org/Ljubljana/RomanJerala"><br />
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<area shape="rect" <br />
coords="217,146,264,334" <br />
alt="Rok Gaber"<br />
href="https://2007.igem.org/Ljubljana/RokGaber"><br />
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<area shape="rect" <br />
coords="329,132,383,330" <br />
alt="Andrej Ondracka"<br />
href="https://2007.igem.org/Ljubljana/AndrejOndracka"><br />
<br />
<area shape="rect" <br />
coords="445,140,499,332" <br />
alt="Peter Cimermancic"<br />
href="https://2007.igem.org/Ljubljana/PeterCimermancic"><br />
<br />
<area shape="rect" <br />
coords="544,52,623,376" <br />
alt="Marko Dolinar"<br />
href="https://2007.igem.org/Ljubljana/MarkoDolinar"><br />
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<area shape="rect" <br />
coords="139,48,180,127" <br />
alt="Gabriela Panter"<br />
href="https://2007.igem.org/Ljubljana/GabrielaPanter"><br />
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<area shape="rect" <br />
coords="182,79,218,196" <br />
alt="Mojca Bencina"<br />
href="https://2007.igem.org/Ljubljana/MojcaBencina"><br />
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<area shape="rect" <br />
coords="220,50,257,120" <br />
alt="Mateja Mancek Keber"<br />
href="https://2007.igem.org/Ljubljana/MatejaMancekKeber"><br />
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<area shape="rect" <br />
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<area shape="rect" <br />
coords="317,64,369,129" <br />
alt="Karolina Ivicak"<br />
href="https://2007.igem.org/Ljubljana/KarolinaIvicak"><br />
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<area shape="rect" <br />
coords="390,45,419,185" <br />
alt="Marko Bitenc"<br />
href="https://2007.igem.org/Ljubljana/MarkoBitenc"><br />
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coords="427,58,463,132" <br />
alt="Anja Korencic"<br />
href="https://2007.igem.org/Ljubljana/AnjaKorencic"><br />
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<area shape="rect" <br />
coords="471,59,518,138" <br />
alt="Katja Kolar"<br />
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<h3><span>Undergraduate Students</span></h3><br />
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<ul><br />
<li><a href="https://2007.igem.org/Ljubljana/MarkoBitenc">Marko Bitenc</a> (Biotechnology, 3rd year)<br />
<li><a href="https://2007.igem.org/Ljubljana/PeterCimermancic">Peter Cimermančič</a> (Biochemistry, 3rd year)<br />
<li><a href="https://2007.igem.org/Ljubljana/RokGaber">Rok Gaber</a> (Microbiology, 4th year)<br />
<li><a href="https://2007.igem.org/Ljubljana/SasaJereb">Saša Jereb</a> (Biochemistry, 2nd year)<br />
<li><a href="https://2007.igem.org/Ljubljana/KatjaKolar">Katja Kolar</a> (Biochemistry, 2nd year)<br />
<li><a href="https://2007.igem.org/Ljubljana/AnjaKorencic">Anja Korenčič</a> (Biochemistry, 4th year)<br />
<li><a href="https://2007.igem.org/Ljubljana/AndrejOndracka">Andrej Ondračka</a> (Biochemistry, 3rd year)<br />
</ul><br />
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<br><br />
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<h3><span>Instructors</span></h3><br />
<p class="p1"><br />
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<ul><br />
<li><a href="https://2007.igem.org/Ljubljana/RomanJerala">Roman Jerala</a> <br />
<li><a href="https://2007.igem.org/Ljubljana/MarkoDolinar">Marko Dolinar</a> <br />
<br><br />
<li><a href="https://2007.igem.org/Ljubljana/MojcaBencina">Mojca Benčina</a><br />
<li><a href="https://2007.igem.org/Ljubljana/KarolinaIvicak">Karolina Ivičak</a> <br />
<li><a href="https://2007.igem.org/Ljubljana/MatejaMancekKeber">Mateja Manček Keber</a> <br />
<li><a href="https://2007.igem.org/Ljubljana/GabrielaPanter">Gabriela Panter</a> <br />
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/summaryLjubljana/summary2007-11-23T18:53:28Z<p>RGaber: </p>
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<link rel="stylesheet" href="/igem07/index.php?title=User:RGaber/stylesheet/stylenew_zadaj.css&action=raw&ctype=text/css" type="text/css" /><br />
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<h3><span>Achievements</span></h3><br />
<p class="p1"><span><br />
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<ul><br />
<li>Design and implementation of three functional systems of activation of anti-HIV defense based on:<br />
<dl><br />
<dd>- detection of viral interactions with the cells and<br />
<dd>- cleavage of specific substrate by viral protease.<br />
</dl><br />
<li>Successful detection of CD4-CCR5 heterodimer formation through: <br />
<dl><br />
<dd>- split tobacco etch virus (TEV) protease,<br />
<dd>- split ubiquitin system.<br />
</dl><br />
<li>Construction of <a href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a> for heterodimerization, which represents a powerful platform for engineering cell processes based on protein-protein interactions.<br />
<li>Introduction of a versatile amplification platform based on the use of T7 RNA polymerase in mammalian cells.<br />
<li>Activation device based on the proteolytic cleavage and release of membrane-anchored T7 RNA polymerase from the membrane into the cell nucleus.<br />
<li>Anchoring of T7 RNA polymerase to the plasma membrane either by:<br />
<dl><br />
<dd>- fusion to CD4 or<br />
<dd>- addition of a myristoylation signal.<br />
</dl><br />
<li>Implementation of two different types of effector molecules for: <br />
<dl><br />
<dd>- induction of cell death (caspase-3) or<br />
<dd>- activation of antiviral defense (interferon beta).<br />
</dl><br><br />
<li>Preparation of a 12-pages <a href="https://static.igem.org/mediawiki/2007/9/99/BrochureSB07_LJU_TitlePage.jpg">brochure on Synthetic Biology</a> aimed at high school students and general audience. <br />
</dl><br />
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</a></span></p><br />
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<h3><span>Prospects for future work and real world applications</span></h3><br />
<p class="p1"><br />
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<br />
<ul><br />
<li>The idea of relying on viral functions could be applied also to other HIV-specific processes (such as genome integration, reverse transcription…).<br />
<li>Our system based on <a href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a> has been useful to prove the principle, but for the real application cellular delivery would be much better applied using vectors based on viral delivery.<br />
<li>In the case of using viral vectors, therapy could be targeted specifically to cells that can be infected by HIV-1 (e.g. similar to T-cell-directed retroviral delivery systems described by David Baltimore at SB2.0).<br />
<li>Selection of different effectors will (and can) be made based on further experiments – apoptosis can only be used in 100% leak-proof systems, while antiviral and immune response effectors are not as sensitive.<br />
<li>Similar systems can be prepared for therapy of many other pathogens, particularly viruses (e.g. yellow fever, SARS, foot and mouth disease…) which contain specific proteases and rely on defined cellular receptors.<br />
</ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
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<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
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<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
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<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<br><br><br />
</div> <br />
<br />
<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/methodsLjubljana/methods2007-11-23T18:52:41Z<p>RGaber: </p>
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<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
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<h3><span>Parts Design</span></h3><br />
<p class="p1"><span><br />
<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td> <img src ="https://static.igem.org/mediawiki/2007/d/da/Parts_design.JPG"> <br />
<small><b> Anja is preparing samples for plasmid isolation. </small></b></td><br />
<td> Since almost all of the parts used by our team had to be designed <i>de novo</i>, a huge portion of our time in the lab was spent on cloning. For each basic part, we designed primers containing standard BioBrick restriction sites. We had to introduce point mutations into some genes, because they contained one or more standard restriction sites inside the coding region. These mutations were introduced by PCR-driven overlap extension method (Heckman and Pease, 2007).<br> <br />
The genes, amplified by PCR, were therefore flanked by <i>Xba</i>I site upstream and <i>Spe</i>I, <i>Not</i>I and <i>Pst</i>I downstream of the gene. After restriction, fragments were cloned into <a href="http://partsregistry.org/Part:BBa_J52017">BBa_J52017</a>, a vector already containing a eukaryotic terminator sequence downstream of the cloning region. This vector has previously been constructed by the Slovenian iGEM 2006 team. </td><br />
</tr><br />
</table><br />
<br />
In addition to basic parts, we also designed dozens of composite parts, mainly constructs expressing chimeric proteins. Composite parts were assembled by three-point ligations, leaving 6 nucleotides long <i>Spe</i>I-<i>Xba</i>I mixed restriction site inbetween the two domains. Some of our composite parts contain as many as 6 basic parts. Such composite parts were constructed by repeating three point ligations.<br />
<br />
</a></span></p><br><br />
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<br />
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<div align="justify" id="development"><br />
<h3><span>Cell cultures</span></h3><br />
<p class="p1"><br />
<br />
<br />
<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td> In our experiments two different cell lines were used: human embrional kidney cell line HEK293T and human cervical cancer cell line HeLa. Cells were grown in DMEM medium containing 10% FBS and antibiotics streptomycin and penicillin, except during transfection when antibiotics were absent from the medium. For luciferase assays, cells were tipically seeded in 96-well plates, for microscopy in slide chambers, whereas for Western blotting and cytometry, where larger numbers of cells were needed, they were grown in 6-well plates, 35 mm dishes or even 10 cm<sup>2</sup> dishes. Depending on the type of experiment Lipofectamine 2000 or GeneJuice were used for transfection according to manufacturer´s protocol. Cells were transfected with the desired plasmids, stimulated if necessary and left grown before detection. </td><br />
<td> <img src ="https://static.igem.org/mediawiki/2007/5/53/Delo_s_kulturami.JPG"><small><b> Katja in cell lab - transfecting our cells. </small></b><br />
</td><br />
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<div align="justify" id="development"><br />
<h3><span>Bioluminescence</span></h3><br />
<p class="p1">Bioluminescence is emission of light, produced by enzymatic reaction. In molecular biology, genes coding for enzymes, which catalize reactions where light is emitted, are used as reporter genes. The most widely used reporter enzymes are luciferases.<br><br />
<br />
In our experiments we used firefly luciferase placed under a specific inducible promoter of interest. The amount of sythesized luciferase was therefore dependent on the activation rate of cells and was semiquantified by measuring emitted light after substrate cleavage. As the number of cells and transfection efficiency vary, the results were normalized by a second reporter luciferase (the <i>Renilla</i> luciferase) placed under a constitutive promoter (expression of which is therefore cell activation independent) and with specificity for a different substrate. The measurements were conducted directly on 96-well plates in a luminometer.<br><br />
<br />
Luciferase assay proved to be our most valuable tool due to the extremely low detection limits. We used this assay to test both our split protein and HIV-protease systems. Cells were transfected with CD4 and CCR5 receptors fused with components of split ubiquitin or split TEV protease systems and reporter constructs. T7 promoter-luciferase reporter construct was used to measure the cellular response to viral attack. HIV surface protein g120 induces cleavage of of T7 RNA polymerase fused with a transmembrane segment and its translocation to nucleus, where it starts to transcribe the reporter gene. </p><br><br />
</div><br />
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<div align="justify" id="development"><br />
<h3><span>Western blotting</span></h3><br />
<p class="p1"><br />
<br />
<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td> <img src ="https://static.igem.org/mediawiki/2007/7/74/Blotanje.JPG"> <small><b> Andrej - specialist for Western blots. </small></b></td><br />
<td>Western blotting is a method of protein transfer from SDS-PAGE (SDS-polyacrylamide gel electrophoresis) gels to nitrocelulose membrane, which enables detection of proteins of interest in cell lysates using specific primary antibodies (pAb) and enzyme-conjugated secondary antibodies (sAb), which bind to pAb. In the first step, transfected cells grown in cell cultures were lysed and the insoluble membrane fraction was removed by centrifugation. Loading buffer containing SDS and a reducing agent was then added to the cleared lysate and the samples were denatured by boiling. Next, samples were applied to PAGE, where proteins were separated according to their molecular weight. Separated proteins were then transferred to a nitrocellulose membrane by electrical current. </td><br />
</tr><br />
</table><br />
<br />
The efficiency of the transfer was determined either by staining the gel with Ponceau dye or by using the pre-stained molecular weight markers, which are visible on the membrane after the transfer. After blotting, the free binding sites on the membrane were blocked by 5% non-fat dry milk in order to prevent unspecific binding of antibodies to the membrane. Then, the nitrocellulose membrane was incubated with pAb for 1 h at room temperature or overnight at 4<sup>o</sup>C with gentle shaking. The membrane was then washed to remove the unbound pAb and incubated with horseradish peroxidase-labelled sAb for 1 h at room temperature with gentle shaking. The signal was finally detected on the film by using a chemiluminescent substrate.<br><br />
Western blotting proved to be a very useful technique for determining whether proteolytic cleavage has occured. In the split ubiquitin assay, CMV-CD4-CUb-GFP and CMV-CCR5-NUb constructs were used. By comparing to molecular weight standards, we could detect whether the GFP was cut off from the transmembrane segment or remained attached to it when using anti-GFP antibodies. This method was also used in the HIV-protease project; the construct CMV-CD4-cleavage site-GFP was used and again GFPs of different sizes were detected when cotransfection with plasmid expressing HIV protease was performed.</p><br><br><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify" id="development"><br />
<h3><span>Confocal microscopy</span></h3><br />
<p class="p1">The basis of fluorescence as a technique is excitation of a fluorophore, which then emits light of a specific wavelenght. The main feature of this technique is its very high sensitivity. Confocal microscopy enables three dimensional detection and allocation of flourescent molecules inside the cells. This technique was used to determine the location of CD4 fused to mCerulean or mCherry inside the cells. Additionally, microscopy of cells after transfection with CD4 and CCR5 receptor constructs fused with split protein systems or HIV-protease cleavage site was used to show that after cell activation (with gp120, pseudoviruses or HIV protease) cleavage of mCerulean or mCherry from the receptor occured. Thus, we can expect that the same occurs when T7 polimerase is fused instead of mCerulean or mCherry.<br> <br />
<br />
In our experiments, cell membranes were stained with SynaptoRed C2 (5 mM) for 5 minutes at 25 μM concentration and endoplasmic reticulums with ER-Tracker Red (1 mM) for 10 minutes at 2,5 μM. Cells were viewed using a confocal microscope with an oil-immersion objective. The 458, 514 (all multi-line Argon laser) and 543 (He-Ne laser) wavelengths were used to excite mCerulean, SynaptoRed C2 and mCherry (or ER-Tracker Red), respectively. </p><br><br />
</div><br />
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<br />
<br />
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<br />
<div align="justify" id="development"><br />
<h3><span>Flow cytometry</span></h3><br />
<p class="p1">Flow cytometry is a method which allows monitoring of cells in a flow stream on the basis of light scattering and fluorescence on single cell level. This method allows measuring of more parameters in a time and determination of a share of cells with a specific characteristic. There is a possibility of wide range of applications, however, we used two: annexin V and 7-AAD stainings for detection of apoptotic cells and antibody staining for detection of surface expression of transmembrane proteins.<br><br />
<br />
Apoptotic cells are characterized by certain morphological changes, one of them being translocation of phosphatidylserine (PS) from inner to the outer layer of plasma membrane bilayer. This change occurs early in the process. Annexin V is a protein, which binds specifically to PS. As this translocation of PS occurs early in the process, Annexin V is a sensitive probe to detect early apoptotic cells. Annexin V can be conjugated to a fluorochrome and thus detected by means of flow cytometry.<br> <br />
<br />
In addition to staining with Annexin V-PE (emission 575 nm), cells were also stained with 7-Amino actomycin (7-AAD, emission 670 nm), a dye that binds to necrotic cells. Staining of cells with both markers allows differentiation between early apoptotic cells, as they stain positive only for annexin, and necrotic cells, which stain positive for 7-AAD or both. This system was also used to present the idea as a whole - by using an effector protein which is an activator of apoptosis (such as caspase-3) as a reporter, binding of the virus to CD4 and CCR5 receptors expressed on cells would lead to their death even before the virus itself is able to replicate. This would then be reflected in positive staining of cells as detected by flow cytometry.<br><br />
<br />
Antibody staining was used for detection of transmembrane receptors CD4, CCR5 and CXCR4 on cell surfaces. For this purpose composite parts with receptors and Myc or HA tags, which flutter on the cell surface were designed. After transfection with plasmids expressing these receptors, cells were incubated with primary anti-tag antibodies, and then with secondary antibodies, which bind to constant, species-specific region of primary antibodies. Secondary antibodies were conjugated with a fluorophores (PE or DyeMer), which emit light at 575 nm and 610 nm, respectively. While using specific anti-Myc or anti-HA tag antibodies only cells transfected with our receptor constructs and not endogenous receptors were detected. </p><br><br />
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<h3><span>ELISA</span></h3><br />
<p class="p1">ELISA (enzyme-linked immunosorbent assay) is an immunological technique which enables semiquantitative determination of the amount of molecules of interest in the cell lysate using specific antibodies. Antibodies are bound to the surface of a microtiter plate and molecules of interest present in the cell lysate bind to them. The amount of bound molecules is detected using another primary antibody conjugated with biotin. Streptavidin conjugated with horse radish peroxidase specifically interacts with biotin. Peroxidase enzymatically cleaves a chromogenic substrate and the absorbance is measured. ELISA was used to determine the amount of caspase-3 protein after the activation of the engineered system with HIV protease, gp120 or pseudoviruses.</p><br><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/biobricksLjubljana/biobricks2007-11-23T18:52:04Z<p>RGaber: </p>
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<h3><span>Biobricks</span></h3><br />
<p class="p1"><span><b><br />
<br />
This year, our team has been the driving force of expanding the registry, creating no less than 77 parts. For our purpose we created, in addition to other parts, biobricks coding for individual domains of multi-domain proteins, involved in cellular signalling and antiviral response. These provide particular enrichment of the registry as they allow endless possibility to readily create novel chimeric proteins.<br />
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<h3><span>Slovenian Team Jewelbox</span></h3><br />
<p class="p1"><span>Following parts were deposited into BioBrick database: </span></p><br><br />
</div><br />
<br />
<br />
<div></div></form><br />
<small><br />
<TABLE class='pgrouptable addborder' style='width: 600px;' cellpadding='0' cellspacing='0' border="0" style="background:#d0d2fb"><TR><TH colspan='2' style='width: 1em' nowrap><A style='color:blue;text-decoration:none;'<br />
href='http://partsregistry.org/Help:Availability_and _usefulness'></A><TH style='width: 7em;'>Name</TH><TH>Type<TH>Description<TH>Length<TH class='blank_col'><br />
<br />
<!-- ROW --><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10080" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10080'>BBa_I712002</A></TD><TD>Coding</TD><TD>CCR5</TD><TD ALIGN='right'>1059</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10005" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10005'>BBa_I712003</A></TD><TD>Coding</TD><TD>CCR5-NUb</TD><TD ALIGN='right'>1194</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10004" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10004'>BBa_I712004</A></TD><TD>Regulatory</TD><TD>CMV promoter</TD><TD ALIGN='right'>654</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10032" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10032'>BBa_I712007</A></TD><TD>Tag</TD><TD>c-Myc-tag</TD><TD ALIGN='right'>30</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10033" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10033'>BBa_I712008</A></TD><TD>Tag</TD><TD>HA-tag</TD><TD ALIGN='right'>30</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10162" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10162'>BBa_I712009</A></TD><TD>Coding</TD><TD>CD4 signal peptide</TD><TD ALIGN='right'>75</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10242" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10242'>BBa_I712010</A></TD><TD>Coding</TD><TD>CD4 sequence without signal peptide</TD><TD ALIGN='right'>1299</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10243" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10243'>BBa_I712011</A></TD><TD>Coding</TD><TD>CUb (split ubiquitin system)</TD><TD ALIGN='right'>135</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10084" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10084'>BBa_I712012</A></TD><TD>Coding</TD><TD>TIR domain of TLR3</TD><TD ALIGN='right'>456</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10068" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10068'>BBa_I712015</A></TD><TD>Other</TD><TD>cleavage site for HIV-1 protease</TD><TD ALIGN='right'>42</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10246" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10246'>BBa_I712016</A></TD><TD>Other</TD><TD>myri + TEVP</TD><TD ALIGN='right'>57</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10050" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10050'>BBa_I712017</A></TD><TD>Coding</TD><TD>Chemokine (CXC motif) receptor 4, fused to N-terminal half of ubiquitin.</TD><TD ALIGN='right'>1191</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10049" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10049'>BBa_I712019</A></TD><TD>Reporter</TD><TD>Firefly luciferase - luciferase from Photinus pyralis</TD><TD ALIGN='right'>1653</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10052" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10052'>BBa_I712020</A></TD><TD>Coding</TD><TD>Homo sapiens caspase 3</TD><TD ALIGN='right'>834</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10200" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10200'>BBa_I712021</A></TD><TD>Coding</TD><TD>Interferon beta</TD><TD ALIGN='right'>564</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10163" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10163'>BBa_I712022</A></TD><TD>Coding</TD><TD>T7 RNA polymerase with NLS</TD><TD ALIGN='right'>2673</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10248" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10248'>BBa_I712024</A></TD><TD>Coding</TD><TD>CD4</TD><TD ALIGN='right'>1374</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10244" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10244'>BBa_I712026</A></TD><TD>Tag</TD><TD>myristoilation site</TD><TD ALIGN='right'>21</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10006" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10006'>BBa_I712027</A></TD><TD>Composite</TD><TD>CMV + CCR5-NUb</TD><TD ALIGN='right'>1854</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10062" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10062'>BBa_I712028</A></TD><TD>Coding</TD><TD>CherryNLS - synthetic construct monomeric red fluorescent protein with nuclear localization sequence</TD><TD ALIGN='right'>733</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10109" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10109'>BBa_I712029</A></TD><TD>Composite</TD><TD>CMV-myc-CXCR4-NUb</TD><TD ALIGN='right'>1887</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10110" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10110'>BBa_I712030</A></TD><TD>Composite</TD><TD>CMV-CXCR4-NUb</TD><TD ALIGN='right'>1851</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10304" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10304'>BBa_I712031</A></TD><TD>Intermediate</TD><TD>CMVp-CD4-HA-CUb</TD><TD ALIGN='right'>2219</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10348" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10348'>BBa_I712033</A></TD><TD>Intermediate</TD><TD>CMVp - myristoilation site</TD><TD ALIGN='right'>681</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10350" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10350'>BBa_I712034</A></TD><TD>Intermediate</TD><TD>CMVp - myristoilation site - HIV protease cleavage site</TD><TD ALIGN='right'>731</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10300" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10300'>BBa_I712035</A></TD><TD>Composite</TD><TD>CMVp-CD4-CUb</TD><TD ALIGN='right'>2175</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10343" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10343'>BBa_I712037</A></TD><TD>Composite</TD><TD>CMVp - CD4 - CUb - mCherry - NLS</TD><TD ALIGN='right'>2914</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10344" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10344'>BBa_I712038</A></TD><TD>Composite</TD><TD>CMVp - CD4 - CUb - GFP</TD><TD ALIGN='right'>2901</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10346" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10346'>BBa_I712039</A></TD><TD>Intermediate</TD><TD>CMVp - HA - CD4 - CUb - GFP</TD><TD ALIGN='right'>2945</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10301" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10301'>BBa_I712040</A></TD><TD>Intermediate</TD><TD>CMVp-CD4-HIVcleavageSite</TD><TD ALIGN='right'>2084</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10319" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10319'>BBa_I712041</A></TD><TD>Composite</TD><TD>CMVp - CD4 - HIV protease cleavage site - mCherryNLS</TD><TD ALIGN='right'>2823</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10324" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10324'>BBa_I712043</A></TD><TD>Composite</TD><TD>CMVp - myristoilation site - HIV protease cleavage site - T7RNAP - NLS</TD><TD ALIGN='right'>3410</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10332" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10332'>BBa_I712044</A></TD><TD>Composite</TD><TD>CMVp - myristoilation site - HIV protease cleavage site - mCherry - NLS</TD><TD ALIGN='right'>1470</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10334" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10334'>BBa_I712045</A></TD><TD>Composite</TD><TD>CMVp - myristoilation site - HIV protease cleavage site - GFP</TD><TD ALIGN='right'>1457</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10321" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10321'>BBa_I712046</A></TD><TD>Composite</TD><TD>CMVp - HA - CD4 - HIV protease cleavage site - GFP</TD><TD ALIGN='right'>2854</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10312" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10312'>BBa_I712048</A></TD><TD>Composite</TD><TD>CMV + CD4 with ha tag + TIR (TLR3)</TD><TD ALIGN='right'>2542</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10241" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10241'>BBa_I712049</A></TD><TD>Composite</TD><TD>CMV + Myc + CCR5 + TIR(TLR3)</TD><TD ALIGN='right'>2183</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10250" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10250'>BBa_I712050</A></TD><TD>Composite</TD><TD>CMV promoter - RBS - T7 RNA polymerase - NLS</TD><TD ALIGN='right'>3340</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10207" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10207'>BBa_I712054</A></TD><TD>Reporter</TD><TD>pT7-mCer</TD><TD ALIGN='right'>772</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10335" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10335'>BBa_I712055</A></TD><TD>Composite</TD><TD>myri - TEVP - mCherry - NLS</TD><TD ALIGN='right'>796</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10336" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10336'>BBa_I712056</A></TD><TD>Composite</TD><TD>myri - TEVP - GFP</TD><TD ALIGN='right'>783</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10338" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10338'>BBa_I712057</A></TD><TD>Composite</TD><TD>myri - TEVP - T7 RNAP - NLS</TD><TD ALIGN='right'>2736</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10339" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10339'>BBa_I712058</A></TD><TD>Composite</TD><TD>CMVp - myri - TEVP - GFP</TD><TD ALIGN='right'>1443</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10340" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10340'>BBa_I712059</A></TD><TD>Composite</TD><TD>CMVp - myri - TEVP - T7 RNAP - NLS</TD><TD ALIGN='right'>3396</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10204" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10204'>BBa_I712064</A></TD><TD>Composite</TD><TD>pT7-IFNbeta</TD><TD ALIGN='right'>616</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10164" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10164'>BBa_I712069</A></TD><TD>Coding</TD><TD>RBS-T7 RNA polymerase with NLS</TD><TD ALIGN='right'>2678</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10251" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10251'>BBa_I712070</A></TD><TD>Composite</TD><TD>T7 promoter - RBS -T7 RNA polymerase with NLS</TD><TD ALIGN='right'>2732</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10071" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10071'>BBa_I712073</A></TD><TD>Device</TD><TD>CMV-Cas3</TD><TD ALIGN='right'>1494</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10177" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10177'>BBa_I712074</A></TD><TD>Regulatory</TD><TD>T7 promoter (strong promoter from T7 bacteriophage)</TD><TD ALIGN='right'>46</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10180" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10180'>BBa_I712076</A></TD><TD>Coding</TD><TD>CMV promoter - Myc tag - CCR5 (shortened)</TD><TD ALIGN='right'>1608</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10182" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10182'>BBa_I712077</A></TD><TD>Coding</TD><TD>TEV protease (N-terminal half)</TD><TD ALIGN='right'>357</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10184" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10184'>BBa_I712078</A></TD><TD>Coding</TD><TD>TEV protease (C-terminal half)</TD><TD ALIGN='right'>375</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10193" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10193'>BBa_I712079</A></TD><TD>Coding</TD><TD>CMV promoter - Myc tag - CCR5 (shortened) - TEV N</TD><TD ALIGN='right'>1973</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10198" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10198'>BBa_I712080</A></TD><TD>Coding</TD><TD>CMV promoter - Myc tag - CCR5 (shortened) - TLR3 TIR</TD><TD ALIGN='right'>2072</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10211" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10211'>BBa_I712082</A></TD><TD>Coding</TD><TD>CMV promoter - CD4 (shortened)</TD><TD ALIGN='right'>1907</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10214" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10214'>BBa_I712083</A></TD><TD>Composite</TD><TD>CMV promoter - CD4 (shortened) - TEV C</TD><TD ALIGN='right'>2290</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10215" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10215'>BBa_I712084</A></TD><TD>Composite</TD><TD>CMV promoter - CD4 (shortened) - TLR3 TIR</TD><TD ALIGN='right'>2371</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10293" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10293'>BBa_I712085</A></TD><TD>Composite</TD><TD>CMV - CD4(shortened) - Cub</TD><TD ALIGN='right'>2048</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10294" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10294'>BBa_I712086</A></TD><TD>Composite</TD><TD>CMV - CD4(shortened) - Cub - CherryNLS</TD><TD ALIGN='right'>2646</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10297" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10297'>BBa_I712087</A></TD><TD>Composite</TD><TD>CMV - CD4(shortened) - Cub - T7polNLS</TD><TD ALIGN='right'>4727</TD><TD class ='blank_col'></TD></FORM><br />
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<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10299" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10299'>BBa_I712088</A></TD><TD>Composite</TD><TD>CMV - CD4(shortened) - Cub - GFP</TD><TD ALIGN='right'>2774</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10306" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10306'>BBa_I712090</A></TD><TD>Composite</TD><TD>CMVp-CD4</TD><TD ALIGN='right'>2034</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10345" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10345'>BBa_I712564</A></TD><TD>Composite</TD><TD>CMV + fLuc</TD><TD ALIGN='right'>2313</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10314" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10314'>BBa_I712666</A></TD><TD>Composite</TD><TD>CMV + Myc + CCR5</TD><TD ALIGN='right'>1755</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10333" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10333'>BBa_I712667</A></TD><TD>Coding</TD><TD>HIV protease</TD><TD ALIGN='right'>303</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10337" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10337'>BBa_I712668</A></TD><TD>Composite</TD><TD>CMV - HIV protease</TD><TD ALIGN='right'>963</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10341" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10341'>BBa_I712669</A></TD><TD>Composite</TD><TD>CMV - mCer</TD><TD ALIGN='right'>1380</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10353" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10353'>BBa_I712670</A></TD><TD>Intermediate</TD><TD>CMV-HA</TD><TD ALIGN='right'>690</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10355" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10355'>BBa_I712671</A></TD><TD>Intermediate</TD><TD>CMV-c-myc</TD><TD ALIGN='right'>690</TD><TD class ='blank_col'></TD></FORM><br />
<br />
<form method="post" action="/r/parts/partsdb/pgroup.cgi?pgroup=iGEM2007&amp;group=iGEM2007_Ljubljana" enctype="multipart/form-data"><br />
<input type="hidden" name="Editing" value="Editing" /><input type="hidden" name="part" value="10363" /><input type="hidden" name="pgroup" value="iGEM2007" /><input type="hidden" name="group" value="igem2007_ljubljana" /><TR><TD class='cell_white'>&nbsp;<TD class='cell_white'>&nbsp;<TD><A class='noul_link' href='view.cgi?part_id=10363'>BBa_I712672</A></TD><TD>Composite</TD><TD>CMV-IFNbeta</TD><TD ALIGN='right'>1224</TD><TD class ='blank_col'></TD></FORM></TABLE></FORM><BR><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/finalsystemLjubljana/finalsystem2007-11-23T18:50:05Z<p>RGaber: </p>
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<h3><span>Split systems based on heterodimerization</span></h3><br />
<p class="p1"><span><br />
After all subsystems were tested to be functional and their expression was optimized, final tests were performed to demonstrate that split-ubiquitin and split TEV protease systems were active. For the system to work there are some crucial steps:<br><br />
<ul><br />
<li>viral protein gp120 or (pseudo)viral particles have to bind to ectodomains of cell surface CD4 and CCR5<br />
<li>functional heterodimers of CD4 and CCR5 have to form<br />
<li>intracellular domains of CD4 and CCR5 have to bring split domains into close proximity <br />
<li>split domains have to reconstitute the functional protein (TEVP or ubiquitin)<br />
<li>T7 RNA polymerase needs to be cleaved off and translocated to the nucleus<br />
<li>T7 RNA polymerase has to transcribe a reporter gene<br />
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Our results demonstrate that split-ubiquitin and split TEVP systems were activated by the HIV coat protein gp120 which causes dimerization of CD4 and CCR5 receptors. Activation was amplified by the T7 RNAP system connected to luciferase reporter. Figures 4 and 5 show normalized luciferase activities of gp120-stimulated cells, nonstimulated and control cells, respectively in the split ubiquitin (Fig. 4) and split TEVP (Fig.5) system.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/2/2e/SASAslika1.jpg" width="633" height="380"><br><br />
<small> <b>Fig. 4. Viral envelope protein gp120 activates the split ubiquitin system.</b> Cells were transfected with CMV–CD4–CUb–T7RNA polymerase, CMV–CCR5–Nub, pT7-fLuc and CMV-rLuc reporter plasmids. 12 hours after transfection cells were activated with gp120 protein for additional 12 hours. Cells were lysed and luciferase activity was measured. The results were normalized for transfection efficiency and cell number using rLuc readings. Addition of gp120 clearly activated the split ubiquitin system as higher amounts of T7 promoter-regulated luciferase activity were measured. Luminescence was observed also in cells which were not stimulated, probably because of small level of spontaneusly dimerized CD4 and CCR5, as we used relatively strong promoter, which may cause high level receptors expression at the plasma membrane. T-test: p<0,01, ***; p<0,005, ****<br />
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<img src="https://static.igem.org/mediawiki/2007/8/86/Sasaslika2.jpg" width="633" height="353"><br><br />
<small><b>Fig. 5. Viral gp120 activates the split TEV protease system.</b> Cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal-TEVprotease site–T7 protease, pT7-fLuc and CMV-rLuc reporter plasmids. 12 hours after transfection cells were activated with gp120 protein for additional 12 hours. Cells were lysed and luciferase activity was measured. The results were normalized for transfection efficiency and cell number using rLuc readings. Addition of gp120 activated split TEV protease system as higher amounts of T7promoter luciferase were measured. Increased luminiscence was also observed in control cells, probably due to the same reason as in split ubiquitin experiment. T-test: p<0,1, *.T-test: p<0,005, ****.</small><br><br><br />
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<b>Interpretation of results:</b><br><br />
Firefly luciferase gene is regulated by the inducible T7 promoter, while renilla luciferase is under constitutive CMV promoter. The difference in luminescence of both luciferases is a consequence of different transfection efficiencies and cell number in different wells. Normalization means that we divide relative luminescence of firefly luciferase by relative luminescence of renilla luciferase. The ratio we get presents differences in promoter activity – the higher the ratio the more active promoter we have.<br><br><br />
<br />
Measurements of luminescence revealed a higher firefly/renilla ratio in cells stimulated with viral protein gp120. A significant difference between stimulated and nonstimulated cells was observed. This is a strong evidence that the engineered device actually works as planned.<br><br><br />
<br />
As seen in Figure 1, we could prove enhanced activation of T7 promoter in gp120 stimulated cells. To verify that the response is due only to T7 RNAP liberated from the ubiquitin fusion (and not e.g. by promoter leaking, spontaneous dimerization of CD4/CCR5, unspecific cleavage of membrane-attached T7 polymerase or unexpected activity of membrane-anchored T7 RNAP, we performed two additional control experiments.<br><br><br />
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Control 1: Cells were transfected with T7 promoter – firefly luciferase vector (and renilla luciferase for normalization standard). Only little background luminescence was detected. Control 2: Cells were transfected with both luciferase constructs and with CD4-CUb-T7 RNAP (control column in Figure 1). Luminescence was low, meaning that neither leaking of T7 promoter nor activity of T7 RNA polymerase on the plasma membrane (or its liberation from the bilayer) are the cause of T7 promoter activation.<br><br><br />
<br />
Luminescence was observed also in non-stimulated cells. This could be due to spontaneous dimerization of CD4 and CCR5, probably because we used a relatively strong promoter which could lead to too many receptor molecules expressed and bound to the plasma membrane.<br><br><br />
<br />
In the comparable experiment just with the split-TEVP attached to CD4 and CCR5 and T7 polymerase attached to the plasma membrane via a myristoyl group and connected to the receptor-split TEVP by a TEVP cleavage site. Results, presented in Figure 2 are highly comparable to those obtained with split ubiquitin, were controlled and interpreted as presented above for the split ubiquitin system.<br><br><br />
<br />
In conclusion, with these experiments we proved that stimulation by HIV viral coat protein activated cellular response through both split-protein devices. We can expect that by replacing luciferase by an effector gene (caspase 3 or ß-interferon) cells would respond by activation of apoptosis or by synthesis of antiviral substances which would prevent the virus from spreading infection to healthy cells.<br><br><br />
<br />
<b>Fluorescent confocal microscopy</b> was used to demonstrate the performance of the split TEV protease system and confirm luciferase data. Cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN and myristoylation signal–TEVprotease site–mCerulean. Fluorescence was detected at the plasma membrane, as expected (Fig. 6C). 22 hours after the transfection cells were stimulated with pseudovirus or gp120 protein and again observed 18 hours later. mCerulean was released from the membrane into cytosol in both cases (Fig. 6A and 6B). Additionally, membrane was stained after the stimulation to confirm the cytosolic localization, respectively (Fig. 6B).<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2007/b/b9/KATJA_slika6.jpg" width="633" height="217"><br><br />
<b><small>Fig. 6. HIV pseudovirus and gp120 protein activate split TEV protease system.</b> HEK293T cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal–TEVprotease site–mCerulean. 22 hours after transfection cells were stimulated with pseudovirus (A) or gp120 protein (B) for additional 18 hours. In both cases the split TEV protease system was activated and mCerulean reporter protein was released from membrane to cytosol. However, in unstimulated cells the system remained inactive and mCerulean was detected mainly at the plasma membrane (C).</small><br><br><br />
<br />
<br />
We proposed that analogously T7 RNA polymerase fused to the receptor instead of mCerulean will be cleaved. Transfection with TEV protease cleavage site fused with T7 RNA polymerase with NLS and T7promoter–mCerulean was performed. Cells were stimulated with pseudovirus or gp120 protein 22 hours after transfection and again observed 18 hours later (Fig. 7). In stimulated cells intense expression of mCerulean was detected (Fig. 8A and 8B)whereas unstimulated cells remained clear (Fig. 7C). The system, using receptors CD4 and CCR5 with truncated cytoplasmic domains was also tested and resulted in similar results.<br><br><br />
<br />
These experiments confirm that when activated, split TEV protease system releases T7 RNA polymerase from the membrane. Consequently T7 RNA polymerase translocates to the nucleus where it transcribes genes under the T7 promoter.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/e/e1/KATJA_slika7.jpg" width="633" height="211"><br><br />
<b><small>Fig. 7. Activated split TEV protease system transcribes genes under the T7 promoter.</b> HEK293T cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal–TEVprotease site–T7 RNA polymerase-NLS and reporter plasmid T7-mCerulean. 22 hours after the transfection cells were stimulated with pseudovirus (A) or gp120 protein (B) for additional 18 hours. In both cases the split TEV protease system was activated, resulting in translocation of T7 RNA polymerase from the plasma membrane to the nucleus. Consequently mCerulean reporter protein was transcribed and detected in cytosol. In unstimulated cells the system remained inactive and no fluorescence at all was observed (C).</small><br><br />
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<h3><span>System based on the activation by HIV protease</span></h3><br />
<p class="p1"><span><br />
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The process of system activation was observed using fluorescent confocal microscopy. Cells were transfected with CMV–CD4–HIV protease cleavage site–mCherry–NLS and optionally cotransfected with HIV protease on pNL4.3 plasmid. In cells without HIV protease fluorescence was detected at the plasma membrane (Fig. 8A), whereas in HIV protease cotransfected cells, fluorescent protein mCherry was released from the membrane and translocated to the nucleus (Fig. 8B). Similar results were obtained when mCherry was substituted with T7 RNA polymerase and additional construct pT7–mCerulean was transfected to detect the T7 RNA polymerase activity. In cells with HIV protease cotransfection, mCerulean was detected in cytosol (Fig. 9B), but no fluorescence at all was observed in the control experiment without of the HIV protease activity (Fig. 9A).<br><br><br />
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The system, using myristoylation signal instead of CD4 to anchor the T7 RNA polymerase to the membrane was also tested and we obtained similar results (data not shown).<br><br><br />
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We can conclude that in the presence of HIV protease, T7 RNA polymerase is released from the membrane and translocates to the nuceleus where it transcribes genes under the T7 promoter.<br><br><br />
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<a href="https://static.igem.org/mediawiki/2007/8/8d/KATJA_fig8.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/8/8d/KATJA_fig8.jpg" width="633" height="316"><br />
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<b><small>Fig. 8. HIV protease cleaves the linker between the membrane anchor and mCherry.</b> HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-mCherry-NLS (A) and additionally with HIV protease plasmid pNL4.3 (B). In cells without HIV protease, membrane localization of mCherry reporter protein was observed (A), but in the presence of HIV protease mCherry-NLS was released to cytosol and translocated to nucleus (B).</small><br><br><br />
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<a href="https://static.igem.org/mediawiki/2007/6/6d/Katja_fig9.jpg"><br />
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<b><small>Fig. 9. T7 RNA polymerase is activated by the cleavage of the linker on the membrane anchor by HIV protease.</b> HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-T7 RNA polymerase-NLS, reporter plasmid T7-mCerulean (A) and additionally with HIV protease plasmid pNL4.3 (B). HIV protease cut off T7 RNA polymerase from the membrane, thus activating it. Consequently mCerulean under the T7 promoter was transcribed and detected in cytosol (B). In the absence of protease the system remained inactive and no fluorescence was observed (A).</small><br />
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<h3><span>Effector protein – caspase-3</span></h3><br />
<p class="p1"><span><br />
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Instead of reporter proteins effector proteins such as caspase-3 were used, which are close to the real application. Caspase-3, a cysteine peptidase, which is the main executioner of the apoptosis. We have selected caspase-3 since we wanted to induce apoptosis in HIV-infected cells so that HIV could not replicate and infect the surrounding cells. Apoptosis prevents any inflammation to the neighboring cells and provides for a "clean removal" of apoptotic cells.<br><br><br />
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Two constructs were prepared for testing the caspase-3 activity: CMV-Cas3 and pT7-Cas3. The first construct was used to test if the caspase-3 is active in transfected cells. The second construct with caspase-3 under the T7 promoter was used as our main effector. Caspase-3 will be transcribed only when active T7 RNA polymerase is present in cytosol or nucleus. This should happen when our devices are activated by HIV virus-induced receptor heterodimerization or by HIV protease, expressed in HIV infected cells.<br><br><br />
<br />
ELISA was used to determine the amount of caspase-3 in cells – this indicates the apoptotic potential of our system. We wanted to show that amount of caspase-3 in cells infected with HIV protease is greater than in non-infected cells (Fig. 10). The amount of caspase-3 had to be carefully monitored since the expression of caspase-3 leads to apoptosis and killing the cells, which we then can not detect anymore. So, timing of ELISA tests was therefore very important.<br><br><br />
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<a href="https://static.igem.org/mediawiki/2007/a/a4/CASPASE_fig10.jpg"><br />
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<b><small>Fig. 10. Caspase-3 expression is enchanced in HIV-protease transfected cells.</b> HeLa cells were transfected with effector gene pT7-Cas3 alone or with effector gene and components of system based on activation by HIV protease activity: CD4 membrane receptor with bound T7 RNA polymerase via HIV protease cleavage site and HIV protease in pNL4.3 vector. ELISA test was made with antibodies against human caspase-3. Significant amount of caspase-3 was detected in nontransfected cells and in cells, transfected with pT7-Cas3 as well. Since ELISA can also detect inactive and endogenous caspase-3 and since certain amount of cells is always in apoptosis, we did not expect extremely low values in these two samples. Significantly elevated levels of caspase-3 were detected in cells, transfected with our system components, meaning that more caspase-3 is transcribed and activated cells after HIV protease cuts off T7 RNA polymerase from transmembrane receptor. In terms of our initial idea this means that HIV entry and HIV protease activity in infected cells could trigger apoptosis and thus prevent replication of virus. T-test: p<0,05, **; p<0,01, ***.</small><br><br><br />
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Flow cytometry using annexin V staining was used for detection of amount of apoptotic cells as a consequence of caspase 3 activity after system activation. We have transfected cells with components of our systems (that have been previously proved as functional) with caspase-3 under T7 promoter as an effector gene. As a positive control we used cells, transfected with CMV-Cas3 construct. Activation of apoptosis varies between different cell lines and also the efficiency between different inducers varies significantly. Although some apoptosis was detected still an additional optimization is needed to obtain reliable results.<br><br><br />
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Instead of caspase-3, which triggers apoptosis in HIV infected cells we could use effector proteins that would act as antiviral agents. Good example is interferon-β. Interferons are very important in antiviral defense. They are usually secreted as a consequence of elevated levels of dsRNA in cells. Its effects include prevention of viral replication and stimulation of the immune response. In extreme cases it can also trigger apoptosis, probably trough increased production of p53 gene product.<br><br><br />
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We have not directly tested the activity of human interferon-β in HIV-infected cells but have deposited BioBricks containing interferon-β, pT7-interferon-β and CMV-interferon-β to the Registry.<br><br><br />
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<h3><span>Subsystems</span></h3><br />
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<h3><span>Surface expression of transmembrane receptors CD4, CCR5 and CXCR4 <br></span></h3><br />
<p class="p1"><span><br />
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<br>Surface expression of transmembrane receptors was measured by <a href="https://2007.igem.org/Ljubljana/methods">flow cytometry</a>.<br />
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We have confirmed expression of the following versions of transmembrane receptors at the surface of cell membrane: CD4(HA)-CUb-GFP, CCR5(c-myc)-NUb and CXCR4(c-myc)-NUb (Fig. 1). Additional constructs with transmembrane receptors were also tested and found to be located at the cell membrane. Expression of transmembrane receptors at the cell surface was crucial for our split systems since it shows appropriate protein folding, thus suggesting that receptors are also functional. Both split-ubiquitin and split-TEV system are activated by heterodimerization of transmembrane receptors after their interaction with viral particles.<br />
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The following experiments were done only with CD4 and CCR5 constructs (used by macrophage-tropic viruses). CXCR4 constructs represent receptors for another group of HIV (lymphotropic or dual-tropic viruses) that utilize CXCR4 receptors for entry into cells. Thus we can expand the range of usefulness of our idea to other viral strains.<br><br />
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<img src="https://static.igem.org/mediawiki/2007/e/ea/Citometerzanawiki.jpg" width="600" height="445"><br><br />
<small><b>Fig. 1. Transmembrane receptor constructs CD4, CCR5 and CXCR4 are expressed at the cell surface.</b> Transmembrane receptors were tagged with peptide tags, which allow detection with labeled antibodies. All versions of CD4 were tagged with HA-tag, CXCR4 and CCR5 were tagged with c-myc-tag at the extracellular side of the receptors. Cells were transfected with different receptor constructs and incubated. Surface expressed receptors were detected by using anti-HA (mouse) and anti-c-myc (rabbit) antibodies, followed by fluorescently labelled secondary antibodies (anti-mouse-PE and anti-rabbit-DyeMer). Receptors were detected by flow cytometry. Fluorophores were excited with 488 argon laser and emitted light was detected at 575 and 610 nm, respectively. 10000 events were collected and results are presented as mean fluorescence values at defined emission wavelenght. In comparison with non-transfected cells we can see that our transmembrane receptors are expressed at the cell surface.</small><br><br />
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<h3><span>T7 promoter (pT7) in mammalian cells <br></span></h3><br />
<p class="p1"><span><br />
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T7 promoter is one of the most important aspects of our system since all our reporter and effector proteins are put under its control. Split-ubiquitin, split TEV and HIV protease systems all release T7 RNA polymerase after viral activated heterodimerization of receptors or HIV protease cutting at specific sequence. T7 RNA polymerase with NLS (nuclear localisation sequence) is translocated into the nucleus. T7 RNA polymerase transcribes genes that are controlled by the T7 promoter (reporter, effector and self-amplifying genes).<br><br />
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We wanted to show that:<br><br />
<ul><br />
<li>T7 RNA polymerase system is specific enough to be active only when the system is activated by the presence of HIV (by viral heterodimerization of receptors or by HIV protease),<br />
<li>T7 RNA polymerase is not active when bound to the cell transmembrane receptors,<br />
<li>T7 RNA polymerase is activated when it is cut off from the transmembrane receptor.<br />
</ul><br />
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We have tested T7 promoter by adding different reporter genes under its control. Constructs pT7-fLuc and pT7-mCer were prepared to test our system with lmeasurement of the uciferase luminiscence and by confocal microscopy.<br><br />
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Luciferase measurements were used to determine an optimal amount of an effector gene under the T7 promoter and the activity of T7 RNA polymerase. The reason for optimization was observation of a small amount of luciferase expression under the T7 promoter even in absence of T7 RNA polymerase when cells were transfected with high amount of the cinstruct under the T7 promoter. With the system consisting of CMV-T7pol and pT7-fLuc in different ratios we have demonstrated that at optimal ratios of pT7-effector gene and T7 RNA polymerase we can guarantee specific expression of effector genes (Fig. 2). In the final stage of our project the luciferase is replaced with an effector gne, such as caspase-3. If T7 RNA polymerase is specifically activated by HIV receptor dimerization or by protease activity, caspase-3 should be expressed only in cells under attack and cause their apoptosis.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/2/2d/ANJA_FIG2.JPG" width="633" height="401"><br><br />
<small><b>Fig. 2. Expression of luciferase under T7 promoter is proportional to the amount of added T7 RNA polymerase.</b> HEK293 cells were transfected with 50 ng of pT7-fLuc construct and increasing amounts of stimuli independent CMV-T7 RNA polymerase. After incubation cells were lysed and the amount of expressed luciferase was measured. 50 ng of pT7-fLuc was low enough to prevent spontaneous transcription from T7 promoter. By adding T7 RNA polymerase we triggered specific transcription of luciferase. Higher amount of added T7 RNA polymerase resulted in stronger activation of the system was observed. T-test: p<0.005, ****.</small><br><br><br />
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To confirm the results with with luciferase reporters additional experiment was performed with construct a pT7-mCer, which is a fluorescent protein. Confocal microscopy showed that in the absence of T7 RNA polymerase mCer is not formed. After addition of T7 RNA polymerase we can detect expression of mCer in the cytosol (data not shown).<br><br><br />
<br />
The next step was to show that T7 RNA polymerase is inactive when bound to the membrane proitein CD4 by a peptide linker containing the HIV protease recognition site (Fig. 3). Cells were co-transfected with different amounts of the following constructs: CD4-HIVpro-T7pol(NLS), pT7-rLuc and HIV protease. In the absence of HIV protease T7 RNA polymerase can not transcribe renilla luciferase gene and luciferase activity is not detectable. After the addition of HIV protease T7 RNA polymerase is released from the membrane anchor. It is activated and stays in the cytoplasm or travels to the nucleus because of its nuclear localization sequence. Genes under the T7 promoter control are transcribed and luciferase activity is observed. Again the reporter gene rLuc is replaced by an effector caspase-3 gene under T7 promoter, which would cause apoptosis only in HIV-infected cells.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/6/60/ANJA_FIG3_koncna.jpg" width="633" height="373"><br><br />
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<small><b>Fig. 3. Transcriptional activity of T7 RNA polymerase is activated by cleavage of the linker to the membrane anchor by HIV protease.</b> HEK293 cells were transfected with reporter plasmid pT7-rLuc, transmembrane receptor with T7 RNA polymerase fused to it by the HIV protease recognition site and different amounts of HIV protease plasmid. In cells without HIV protease only weak luciferase activity was observed. Addition of HIV protease cleaved the linker and activated T7 RNA polymerase, which in return transcribed the luciferase under the T7 promoter. T-test: p<0,1, *.</small><br><br><br />
In our model we have proposed a construct pT7-T7pol, which would increase the number of active T7 RNA polymerases in a positive autoloop since entry of HIV into cells or HIV protease can only activate a small amount of T7 RNA polymerase. If we want to have fast and strong effector protein action we must guarantee large amount of active T7 polymerase as soon as possible. Construct pT7-T7pol has been made and deposited into BioBrick database.<br><br />
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<h3><span>Results</span></h3><br />
<p class="p1"><b>Before testing the full functional devices we had to do some research on individual BioBricks to prove their expression and activity of their protein products in mammalian cell cultures. Preliminary testing included control of surface expression of transmembrane receptors with flow cytometry and testing and optimization of the T7 promoter system in mammalian cells.<br><br />
<br />
We continued with testing of individual subsystems; fluorescent reporter proteins were used to prove the functionality of subsystems with confocal microscopy and luminescence for luciferase reporters. Western blotting was used to proteolytic cleavage of test split and TEV protease subsystems. Final stage was testing of effector protein activity – caspase 3. Its expression was determined by ELISA test and its activity, resulting in cell apoptosis was detected by flow cytometry.<br><br />
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We have shown that anti-HIV defense based on HIV entry or its activity in infected cell can lead to transcription and activation of effector proteins that either trigger apoptosis or enhance antiviral immune response.<br><br />
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Detailed information on results of our experiments can be found in the following chapters.<span></b><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/modelLjubljana/model2007-11-23T18:47:26Z<p>RGaber: </p>
<hr />
<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
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<div align="justify" id="aboutred"><br />
<h3><span>Model</span></h3><br />
<p class="p1"><span><b>We built a theoretical model to show how the positive feedback loop made of T7 RNA polymerase (T7 RNAP) gene under the control of its T7 promoter affects the behaviour of the system, particularly amplification. Initially, active T7 RNAP molecules are generated by any of the three sources: split-ubiquitin reconstitution and endogenour ubiquitin protease, TEV protease reconstitution or cleavage by the HIV protease. In all cases, T7 polymerase translocates into the nucleus, where it transcribes an effector gene, as well as T7 RNAP gene (self-amplification). The idea behind introducing such a positive feedback loop was that the initial signal might be too low and could fade if not amplied to a sufficient level.</b></span></p><br />
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</div><br />
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</div><br />
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<br />
<br />
<br />
<br />
<br />
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<br />
<br />
<div id="supportingText"><br />
For modelling interactions and enzymatic reactions, Michaelis-Menten kinetics for one or two substrates was used.<br />
<br />
<br><br><br />
<br />
<br />
<br />
<div id="products"><br />
<h3><span>Model built in Cell Designer</span></h3><br />
<p class="p1"><span><br />
<br />
<a href="https://static.igem.org/mediawiki/2007/6/6f/Modelslika1.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/6/62/Model_low.jpg" width="650" height="488"><br />
</a><br><br />
<small><b>Figure 1:</b> Comprehensive view of the engineered pathway. Split ubiquitin pathway is shown at the lower half and HIV protease at the upper half. HIV protease synthesis pathway after the infection is simplified in this model.</small><br />
</p></div><br />
<br><br />
<br />
<br />
<br />
<div align="justify" id="support"><br />
<h3><span>Signal Amplification</span></h3><br />
<p class="p1"><br />
<br />
Plots of the amount of the active T7 RNAP (red line) and effector (blue line) versus time. The infection begins at time 0. Parameters are selected arbitrarily and the simulation should be used to evaluate the behavior of the system under different gene ratios:<br><br><br />
<br />
<table cellspacing="0" cellpadding="0" border="0" style="border-collapse: collapse; background:#d0d2fb" class="MsoTableGrid"><br />
<tbody><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<a href="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2a)</b> Simulation with 2 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small><br />
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</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2b)</b> Simulation with 50 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small><br />
<br />
</o:p></p><br />
</td><br />
</tr><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2c)</b> simulation with 2 copies of the effector gene and 2 copies of T7 RNAP genes present in the nucleus (the red line is hidden under the green line because of the same transcription and translation kinetics parameters).</small><br />
<br />
</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2d)</b> simulation with 50 copies of the effector and 2 copies of T7 RNAP genes present in nucleus.</small><br />
<br />
</o:p></p><br />
</td><br />
</tr><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2e)</b> simulation with 2 copies of the effector and 50 copies of T7 RNAP genes present in nucleus.</small> <br />
<br />
</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
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</tbody><br />
</table><br />
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<br />
<br><br />
As expected, in case <b>(a)</b>, where no additional amplification was designed, the amount of the effector increases linearily with time. With more copies of the effector gene present, the slope increases <b>(b)</b>. In case <b>(c)</b> with amplification through T7 RNAP, we were expecting the effector amount to grow exponentially. However, contrary to simple reasoning, this is not the case unless the number of effector gene copies is much larger than the number of T7 RNAP gene copies. This can be explained by a limiting rate of transcription; the maximal transcription rate is determined by how many polymerase molecules can bind to the promoter in a certain span of time. Therefore, if the number of polymerase molecules is already high, the system becomes saturated very soon and any further increase of polymerase concentration does not increase expression rate, so no exponential growth of effector concentration occurs. However, if we increase the number of effector gene copies <b>(d)</b>, the system does not readily become saturated and we observe an exponential growth of effector concentration. After a certain time, the number of polymerase molecules increases, and effector genes become saturated. This again results in a linear growth of effector concentration.<br><br />
<br><br />
The model clearly showed that in order to successfully amplify the initiatial signal, it is optimal to use a much larger number of effector genes than of T7 RNAP genes.<br><br />
<br />
</a></span></p><br><br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
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</ul><br />
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<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
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<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
<br />
<br />
</ul><br />
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/modelLjubljana/model2007-11-23T18:46:42Z<p>RGaber: </p>
<hr />
<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
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<br />
<br />
<br />
<br />
<br />
</div><br />
<div align="justify" id="aboutred"><br />
<h3><span>Model</span></h3><br />
<p class="p1"><span><b>We built a theoretical model to show how the positive feedback loop made of T7 RNA polymerase (T7 RNAP) gene under the control of its T7 promoter affects the behaviour of the system, particularly amplification. Initially, active T7 RNAP molecules are generated by any of the three sources: split-ubiquitin reconstitution and endogenour ubiquitin protease, TEV protease reconstitution or cleavage by the HIV protease. In all cases, T7 polymerase translocates into the nucleus, where it transcribes an effector gene, as well as T7 RNAP gene (self-amplification). The idea behind introducing such a positive feedback loop was that the initial signal might be too low and could fade if not amplied to a sufficient level.</b></span></p><br />
<br />
<br />
</div><br />
<br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="supportingText"><br />
For modelling interactions and enzymatic reactions, Michaelis-Menten kinetics for one or two substrates was used.<br />
<br />
<br><br><br />
<br />
<br />
<br />
<div id="products"><br />
<h3><span>Model built in Cell Designer</span></h3><br />
<p class="p1"><span><br />
<br />
<a href="https://static.igem.org/mediawiki/2007/6/6f/Modelslika1.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/6/62/Model_low.jpg" width="650" height="488"><br />
</a><br><br />
<small><b>Figure 1:</b> Comprehensive view of the engineered pathway. Split ubiquitin pathway is shown at the lower half and HIV protease at the upper half. HIV protease synthesis pathway after the infection is simplified in this model.</small><br />
</p></div><br />
<br><br />
<br />
<br />
<br />
<div align="justify" id="support"><br />
<h3><span>Signal Amplification</span></h3><br />
<p class="p1"><br />
<br />
Plots of the amount of the active T7 RNAP (red line) and effector (blue line) versus time. The infection begins at time 0. Parameters are selected arbitrarily and the simulation should be used to evaluate the behavior of the system under different gene ratios:<br><br><br />
<br />
<table cellspacing="0" cellpadding="0" border="0" style="border-collapse: collapse; background:#d0d2fb" class="MsoTableGrid"><br />
<tbody><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<a href="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2a)</b> Simulation with 2 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small><br />
<br />
<br />
</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2b)</b> Simulation with 50 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small><br />
<br />
</o:p></p><br />
</td><br />
</tr><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2c)</b> simulation with 2 copies of the effector gene and 2 copies of T7 RNAP genes present in the nucleus (the red line is hidden under the green line because of the same transcription and translation kinetics parameters).</small><br />
<br />
</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2d)</b> simulation with 50 copies of the effector and 2 copies of T7 RNAP genes present in nucleus.</small><br />
<br />
</o:p></p><br />
</td><br />
</tr><br />
<tr style=""><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
<br />
<br />
<a href="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg"><br />
<img border="0" src="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg" width="313" height="211"><br />
</a><br><br />
<small><b>Figure 2e)</b> simulation with 2 copies of the effector and 50 copies of T7 RNAP genes present in nucleus.</small> <br />
<br />
</o:p></p><br />
</td><br />
<td width="307" valign="top" style="padding: 0cm 5.4pt; width: 230.3pt;"><br />
<p class="MsoNormal"><o:p><br />
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As expected, in case <b>(a)</b>, where no additional amplification was designed, the amount of the effector increases linearily with time. With more copies of the effector gene present, the slope increases <b>(b)</b>. In case <b>(c)</b> with amplification through T7 RNAP, we were expecting the effector amount to grow exponentially. However, contrary to simple reasoning, this is not the case unless the number of effector gene copies is much larger than the number of T7 RNAP gene copies. This can be explained by a limiting rate of transcription; the maximal transcription rate is determined by how many polymerase molecules can bind to the promoter in a certain span of time. Therefore, if the number of polymerase molecules is already high, the system becomes saturated very soon and any further increase of polymerase concentration does not increase expression rate, so no exponential growth of effector concentration occurs. However, if we increase the number of effector gene copies <b>(d)</b>, the system does not readily become saturated and we observe an exponential growth of effector concentration. After a certain time, the number of polymerase molecules increases, and effector genes become saturated. This again results in a linear growth of effector concentration.<br><br />
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The model clearly showed that in order to successfully amplify the initiatial signal, it is optimal to use a much larger number of effector genes than of T7 RNAP genes.<br><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/implementationLjubljana/implementation2007-11-23T18:45:02Z<p>RGaber: </p>
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<h3><span>Implementation</span></h3><br />
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<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
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<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
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<h3><span>Split ubiquitin system</span></h3><br />
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For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
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It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
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T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
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When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
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<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="https://static.igem.org/mediawiki/2007/8/86/Split_ubiquitin.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="https://static.igem.org/mediawiki/2007/8/86/Split_ubiquitin.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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Animation of split-ubiquitin system.<br><br />
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<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
<br />
For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
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<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="https://static.igem.org/mediawiki/2007/0/0a/Split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="https://static.igem.org/mediawiki/2007/0/0a/Split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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Animation of split-TEV protease system.<br><br><br />
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<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
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Animation of HIV protease system.<br><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/StrategyLjubljana/Strategy2007-11-23T18:41:52Z<p>RGaber: </p>
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<h3><span>Strategy</span></h3><br />
<p class="p1"><span><b><br />
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The HIV virus mutates at a rate of 3x10<sup>-5</sup> per nucleotide per replication cycle and with generation of 10<sup>9</sup> to 10<sup>10</sup> virions every day it is clear that viral mutability has to be bypassed in order to develop an efficient antiviral therapy.<br />
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<h3><span>Requirements for the effective antiviral treatment:</span></h3><br />
<p class="p1"><span><br />
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<ul><br />
<li>should be <b>BASED ON VIRAL FUNCTION</b> and <b>INSENSITIVE TO MUTATIONS</b><br />
<li>should be <b>VERSATILE</b> - allow activation of different effectors, e.g.<br />
<dl><br />
<dd>- those that kill infected cells that spread infection (activation of apoptosis)<br />
<dd>- destroy virus (RNase, APOBEC3...)<br />
<dd>- activate human immune defense system (e.g. interferons, chemokines…)<br />
</dl><br />
<li>system should be able to respond to low levels of activation – the <b>RESPONSE SHOULD BE AMPLIFIED</b><br />
<li>avoid activation in noninfected cells<br />
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<h3><span>Considered approaches</span></h3><br />
<p class="p1"><br />
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<ul><br />
<li>use of siRNA - attractive but <b>not suitable because of the mutations</b><br />
<li>antibodies (same problem)<br />
<li>use of viral LTR promoter - experimental evidence that transcription is "leaky"<br />
<li>use the chimeras with TLR3 intracellular domain, which activates the interferon beta signaling pathway - problem with active homodimers <font color="red">(tested, high constitutive activity)</font><br />
<li>use viral reverse transcriptase to integrate the effector into the genome (difficult, perhaps next time :-) )<br />
<li>use of viral protease activity <font color="green">(OK, works)</font><br />
<li>detect formation of CD4-CCR5/CXCR4 heterodimer <font color="green">(OK, works)</font><br />
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<h3><span>Components of the antiviral defense device</span></h3><br />
<p class="p1"><b>Detection / Activation</b><br><br />
We decided to concentrate on two viral functions - binding to cellular receptors and processing of viral proteins by HIV protease.<br><br />
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For detection of viral binding to the cells we took advantage of the formation of the CD4 and CCR5 (or CXCR4) coreceptor dimer upon binding of viral gp120 protein. We looked for ways to detect the formation of heterodimeric membrane proteins and couple this event to the cellular response. We found that the split ubiquitin system has been previously used in yeast to detect the interaction between membrane proteins (Stagljar and Fields, 2002), which is exactly what we needed, except that its function has not yet been described for mammalian membrane proteins. To be safe, the other, parallel approach was to use the split TEV (tobacco etch virus) protease system (Wehr et al., 2006), which restores the proteolytic activity upon heterodimerization of their fusion partners.<br />
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HIV protease activation could be detected by specific cleavage of a protein containing a linker with a protease substrate site similar to the one previously reported before by introduction of a substrate protein into HIV-infected cells (Vocero-Akbani et al, 1999).<br><br><br />
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<b>Amplification</b><br><br />
Detection of infection of a cell with a single viral particle should provoke a significant response, therefore the signal would have to be amplified. This can be achieved by biological processes which employ different enzymes (e.g. activation of a prodrug by reconstituted lactamase...) or through coupling to cellular transcription/translation machinery by different RNA polymerases. We have also considered membrane anchoring of DNAse linked to a nuclear localization signal. After cleavage off the membrane, as a result of activation, DNase would migrate into the nucleus and trigger apoptosis. We opted for the T7 RNA polymerase as it specifically binds to the specific promoter sequence and is commonly used in biotechnological applications for bacterial overexpression of proteins. Additionally its functionality in mammalian cells has also been described (Lieber et al, 1989). The response of this amplification mechanism can even be enhanced by the addition of an autoamplifiable BioBrick with positive autofeedback, which thus increases the amount of available T7 RNA polymerase. The switch to turn on the T7 RNA polymerase was achieved by anchoring it to the membrane and adding the NLS signal to direct it to the nucleus after the activation step. The activation step is reached after receptor dimerization, which results in proteolytic degradation of the linker between the membrane anchor and T7 polymerase.<br><br><br />
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<b>Activation of defense effector</b><br><br />
What type of response do we want the cells to have upon infection by a virus?<br><br />
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One possibility is to simply kill the cell, thereby preventing the virus from forming hundreds of copies and spreading the infection. Other possibilities include activation of an organism's defense system using e.g. interferons, or enzymes that destroy viral components, such as APOBEC3, RNase and others. Activation of the defense system by caspases can only be used in leak-proof systems and is probably most effective when it is already too late for other types of defense. It is a great advantage if the same platform can be used to deploy different types of effectors -- this is exactly what systems controlled by T7 promotor provide. Similarly, transcription factors from other cell types (e.g. yeast) could be used as well, as long as they provide the specific responsiveness to activation, resulting from the viral detection.<br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/CurrenttreatmentLjubljana/Currenttreatment2007-11-23T18:41:09Z<p>RGaber: </p>
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<h3><span>Current Disease treatment</span></h3><br />
<p class="p1"><span><b>HIV is a typical retrovirus that upon entry into the host cell reverse transcribes its RNA to DNA, which integrates into the host genome. The viral genes are expressed as polyprotein that is cleaved into functional proteins by HIV protease. Viral coat is composed of lipids and two predominant proteins, gp41 and gp120, which are essential for binding onto the host cell receptor, CD4. Several drugs were developed which target crucial stages of HIV entry into the host cell, its reverse transcription, integration and processing of the polyprotein. Current standard therapy consists of taking a combination of at least three different antiretroviral drugs.</b></span></p><br />
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<h3><span>Viral Life Cycle</span></h3><br />
<p class="p1"><span>HIV (human immunodeficiency virus) is a retrovirus, Its genome is composed of two single stranded RNA molecules. It has a <i>gag/pol/env</i> organization; <i>gag</i> genes (group specific antigen) code for structural proteins, <i>env</i> for proteins that build viral envelope, while <i>pol</i> genes are responsible for viral reproduction (they contain genes for reverse transcriptase, integrase and HIV protease).<br><br><br />
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The HIV envelope consists of lipids and viral glycoproteins gp120 and gp41, which are crucial for binding of HIV to the host cell membrane and for entering into the cell. Inserted into the lipid bilayer are also other glycoproteins that guarantee firmness and protective function of the viral envelope. Gp120 binds to receptors (CD4) on the host cell surface, but additional co-receptors like chemokine receptors (CCR5, CxCR4) are also required for successful entry of HIV. Mutations in co-receptor genes can cause immunity – if HIV cannot enter host cells, HIV infection is prevented, and AIDS cannot develop.<br><br><br />
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The characteristic retroviral enzyme is reverse transcriptase, which transcripts viral RNA into DNA. Only DNA can integrate into the host cell genome – this is the crucial step in expressing viral proteins that are needed for assembly of new viral particles. Viral <i> gag </i>and <i>gag/pol</i> genes are expressed as polyprotein; until this polyprotein is cut into functional units, it exerts no biological function. Polyprotein clipping is done by HIV protease. The resulting polyprotein fragments represent functional enzymes and structural proteins.<br><br><br />
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Transcription of viral RNA into DNA and processing of the viral polyprotein are the two most important steps in the HIV replication cycle. These are thus obvious targets for HIV therapeutics. Inhibitors of reverse transcriptase and HIV protease are currently used to treat acute HIV infection.</a></span></p><br><br />
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<h3><span>Current Disease Treatment</span></h3><br />
<p class="p1">One of the first AIDS therapeutics were nucleotide or nucleoside analogues (NRTI – nucleoside-analogue reverse transcriptase inhibitors) – pseudosubstrates, which are during reverse transcription integrated into viral DNA instead of nucleosides during reverse transcription and thus block the transcription. These drugs were superseded by non-nucleoside inhibitors (NNRTI) that could inhibit reverse transcriptase by binding into the allosteric site of the enzyme. The third type of drugs are a family of HIV protease inhibitors. In most cases specific inhibitors are very similar to protease substrates - the only difference is that because they cannot be cut, they block the active site by remaining bound into it.<br><br><br />
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The weakness of all these therapeutics is that they are very sensitive to HIV mutations – HIV can easily mutate and thus become drug resistant. A combination of drugs is used to minimize HIV's potential to develop resistance to each individual drug in the mixture. Some of the drugs induce mutations that have negative effect on the virulence and such drugs can be used in spite of developed resistance.<br><br><br />
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We still do not have a cure for AIDS that would be insensitive to HIV mutations. Our project presents new ways of potential AIDS therapeutics. Our approaches can be considered independent of HIV mutations. We have set up a few of biological ambushes; if HIV manages to avoid them, we presume that it would not be able to infect the cell anyway.</p><br />
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<h3><span>Classes of Antiretroviral Drugs</span></h3><br />
<p class="p1">Antiretroviral drugs are mostly inhibitors of different stages in the HIV life cycle. They are targeted at different enzymes or events that are typical for HIV infection – entry of the virus into the cell, reverse transcription, polyprotein cleavage... and are divided into seven main classes (Drugs Used in the Treatment of HIV Infection, 2007):<br><br><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Type of inhibitor</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Action/target</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Role in current AIDS treatment</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">HIV protease inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">Inhibition of the viral protease which has high sequence specificity. Most inhibitors mimic cleavage sites in target protein chains. Without proteolytic processing viral polyprotein is not able to form new viral particles.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">There are around ten FDA approved drugs that are protease inhibitors (Sequinavir, Indinavir, Nelfinavir...).</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Reverse transcriptase inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">If RNA is not transcribed into DNA, HIV is not able to replicate itself. There are two main types – nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI).</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">Zidovudine was the first FDA approved antiretroviral drug for AIDS treatment. There are numerous other drugs of this class on the market now.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Integrase inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">Inhibition of the viral integrase prevents integration of the transcribed DNA into the host cell genome.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">No drug of this type has been approved yet but there are a few undergoing testing.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Fusion inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">They bind to gp41 and prevent viral fusion with the cell membrane.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">Enfurvitide is used with patients with multi-drug resistant HIV strains.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Entry inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">They compete for binding sites of gp120 receptors – when inhibitor is bound, gp120 cannot bind to receptor and co-receptor, so HIV entry is blocked - e.g. chemokines (SDF-1), AMX3100...</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><strong><span lang="EN-US" style="font-family: Arial;">Other inhibitors</span></strong><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<p class="MsoNormal"><span lang="EN-US" style="font-size: 10pt; font-family: Arial;">Some other inhibitors are targeted at blocking the conversion of polyprotein into mature capsid protein, others are a combination of different types if inhibitors.</span><span style="font-family: Arial;"><o:p></o:p></span></p><br />
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<h3><span>HAART</span></h3><br />
<p class="p1">Current AIDS treatment is based on HAART – highly active antiretroviral therapy. It is a mixture of at least three different antiretroviral drugs. By disturbing the viral replication cycle at different stages it is much harder for HIV to mutate and become drug-resistant. Synergistic enhancers are added; these are drugs that are either inappropriate for monotherapy or are enhancers of metabolism of other antiretroviral drugs. Although HAART can provide a longer and better life for patients with AIDS, it cannot totally eliminate HIV from a patient's body because it cannot excise latent viral DNA that is already integrated into the host genome and therefore has to be taken for life.<br><br><br />
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There are two more problems with HAART – its side effects and cost. We can try to minimize the side effects but we will probably never find a cure for AIDS that would be completely harmless. Because HAART must be taken continuously, its price makes it impossible to use widely in areas with the highest percentage of infected population. Ideally, vaccination could limit the AIDS epidemic because vaccines usually cost less than drugs for continuous treatment and could be affordable for developing countries. After many years of intense research we still do not have a vaccine for HIV and because of its high mutation rate HIV is a difficult target.</p><br />
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<h3><span>Alternative Therapies Under Development</span></h3><br />
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<li>siRNA targeted against virus (LTR, Tat, integrase...) or host proteins (knock down of surface receptors...)<br />
<li>gene therapy - decrease of the surface cellular receptors, addition of decoy soluble receptors<br />
<li>intrabodies, ribozymes directed against viral RNA...<br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/HIVbacgroundLjubljana/HIVbacground2007-11-23T18:40:20Z<p>RGaber: </p>
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<h3><span>HIV-1 Infection Background</span></h3><br />
<p class="p1"><span><b>HIV primarily infects cells that are important in the human immune response: T-cells, macrophages and dendritic cells. By replicating itself in T-cells it destroys them and thus weakens the immune system.</b></span></p><br />
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Primary infection is the first step in the course of HIV infection. After HIV enters the host cell, it starts replicating itself intensively and is widely disseminated throughout the body, with the lymphoid organs becoming seeded with the virus. There is a significant drop in CD4 T cells at this early time. Flu-like symptoms are common and 50 - 75% of patients develop this acute mononucleosis-like syndrome.<br><br><br />
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Approximately one to three months after infection a specific immune response develops, causing the number of viral particles to decrease and the CD4 cell count to rebounce. However, the immune response is unable to clear the infection completely and HIV-infected cells persist in the lymph nodes as viral DNA integrated in the host genome. This period of clinical latency may last for as long as 10 years, but there is still a a high level of ongoing viral replication.<br><br><br />
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Eventually clinically apparent diseases develop such as neoplasms or opportunistic infections. These are a consequence of rapid viral proliferation in CD4 infected cells and their subsequent destruction which weakens the immune system. The onset of these symptoms is known as the progression to AIDS.<br><br><br />
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<td align="justify"> Despite enormous efforts to find the ultimate cure for AIDS there is <b>no efficient treatment that could totally eliminate HIV</b>. The main reason for not being able to develop a useful drug is the lack of proofreading replication enzymes in HIV. Consequently, mutations occur changing the properties of HIV molecules. Thus, resistance evolves quickly, making many existing drugs useless. Nevertheless, certain therapeutics and drug cocktails can slow down AIDS progression and provide a better life for those infected with HIV. Another problem in AIDS treatment is the price of therapeutics; only a fraction of the infected population can afford the expensive treatment.<br><br> </td><br />
<td> <object width="425" height="355"><param name="movie" value="http://www.youtube.com/v/RO8MP3wMvqg&color1=0xd6d6d6&color2=0xf0f0f0&border=0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/RO8MP3wMvqg&color1=0xd6d6d6&color2=0xf0f0f0&border=0" type="application/x-shockwave-flash" wmode="transparent" width="354" height="296"></embed></object><br><br />
<b>Video:</b> Targeting HIV replication. </td><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/AIDSplagueLjubljana/AIDSplague2007-11-23T18:38:34Z<p>RGaber: </p>
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<h3><span>AIDS Epidemics</span></h3><br />
<p class="p1"><span><b>AIDS (Acquired ImmunoDeficiency Syndrome) is one of the most striking problems of the modern world. It is a consequence of HIV (Human Immunodeficiency Virus) infection which attacks and damages the immune system. During the progression of the disease patients become more sensitive to different opportunistic infections which can be lethal. Since it was recognized in 1981, AIDS has killed more than 25 million people.</b></span></p><br />
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According to WHO (World Health Organization) AIDS claims around 3 million lives per year; more than 40 million are believed to be living with AIDS and even more are infected with HIV. The most critical is Sub-Saharan Africa where around 30 million people are believed to be infected with HIV which is more than 60% of all HIV infections. In countries with the highest AIDS prevalence life expectancy is 6,5 years less than it would be without the AIDS pandemic.<br><br><br />
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In the last few years great progress has been made in antiretroviral therapy, but a cure for AIDS is still a dream. HAART (highly active antiretroviral therapy) is efficient in easing the symptoms of AIDS and reducing death rate where this expensive therapy is available to AIDS patients. This means that the life expectancy has risen but the number of HIV infected individuals is increasing.<br><br><br />
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Antiretroviral therapy is not perfect; it cannot completely eliminate HIV from infected cells. Apart from that, it is sensitive to HIV mutations; since HIV mutates rapidly, a lot of therapeutic are useless on mutated HIV strains.<br />
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<b>Adult HIV prevalence at the end of 2005.</b> More than 60 % of infected individuals live in Sub-Saharan Africa. South and South-East Asia (especially India) have more than 15 % of the population infected with HIV. (www.wikipedia.org)<br />
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<p class="p1"><span><b>Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.</b></span></p><br />
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HIV-1 virus is one of the most difficult targets for therapy because it hijacks the cells of our immune system and particularly because the virus mutates rapidly making it drug resistant. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously. <br />
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We propose a different strategy, where we target a specific <b>FUNCTION</b> of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.<br />
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The effect of mutations can thus be avoided since those mutations that cause the loss of the function also render the virus harmless. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on the viral maturation. In our system activation of any of them activates the antiviral cell defense or alternatively kills the infected cells, preventing further spread of infection. The same approach could be implemented for defense against other viral infections.<br />
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We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. <b>Two essential viral functions</b> have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease.<br />
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Binding of virus to human T-cells causes formation of CD4-CCR5 heterodimers, which in our system reconstitutes the split ubiquitin. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of <b>versatile effector genes</b>, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same viral function was successfully utilized in the implementation of the split TEV protease system.<br />
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The second implementation of this idea was to utilize the activity of HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 RNA polymerase. T7 RNA polymerase released from the membrane subsequently activates the defense similar to that described with the split protein system. <b>All three systems work in human cells.</b> We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.<br />
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<p class="MsoNormal"><o:p><b>PLEDGE:</b> All experimental work on this project was performed from May to October 2007 by the undergraduate students participating in the team under the tutorial of instructors. All the students participated at iGEM for the first time.</o:p></p><br />
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<td><a href="http://www.ki.si/">National Institute of Chemistry</a> <br><br />
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<td><br><a href="http://www.uni-lj.si/en">University of Ljubljana</a> <br><br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
<br />
<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<br><br><br />
</div> <br />
<br />
<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
<center><br />
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</body><br />
</html></div>RGaberhttp://2007.igem.org/wiki/index.php/LjubljanaLjubljana2007-11-23T18:30:29Z<p>RGaber: </p>
<hr />
<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
<link href="https://2007.igem.org/User:RGaber/stylesheet/stylenew.css" rel="stylesheet" type="text/css" /><br />
</head><br />
<body><br />
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<div id="container"><br />
<div id="intro"><br />
<div id="pageHeader"><br />
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<div id="naslov"><br />
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<center><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2007/7/73/VIROTRAP.jpg" width="633" height="200"><br />
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</center><br />
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</div><br />
<div align="justify" id="about"> <br />
<p class="p1"><span><b>Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.</b></span></p><br />
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</div><br />
</div><br />
<div id="supportingText"><br />
<div id="products"><br />
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<br />
<br />
<h3><span>Abstract for nonspecialists</span></h3><br />
<div align="justify"><br />
HIV-1 virus is one of the most difficult targets for therapy because it hijacks the cells of our immune system and particularly because the virus mutates rapidly making it drug resistant. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously. <br />
<br />
We propose a different strategy, where we target a specific <b>FUNCTION</b> of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.<br />
<br />
The effect of mutations can thus be avoided since those mutations that cause the loss of the function also render the virus harmless. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on the viral maturation. In our system activation of any of them activates the antiviral cell defense or alternatively kills the infected cells, preventing further spread of infection. The same approach could be implemented for defense against other viral infections.<br />
</div><br />
<br><br><br />
<br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="https://static.igem.org/mediawiki/2007/8/86/Split_ubiquitin.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="https://static.igem.org/mediawiki/2007/8/86/Split_ubiquitin.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
Animation of split-ubiquitin system.<br />
<br />
<br><br><br />
<h3><span>Scientific abstract</span></h3><br />
<div align="justify"><br />
We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. <b>Two essential viral functions</b> have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease.<br />
<br />
Binding of virus to human T-cells causes formation of CD4-CCR5 heterodimers, which in our system reconstitutes the split ubiquitin. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of <b>versatile effector genes</b>, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same viral function was successfully utilized in the implementation of the split TEV protease system.<br />
<br />
The second implementation of this idea was to utilize the activity of HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 RNA polymerase. T7 RNA polymerase released from the membrane subsequently activates the defense similar to that described with the split protein system. <b>All three systems work in human cells.</b> We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.<br />
</div><br />
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<br />
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<table cellspacing="0" cellpadding="0" border="1" style="border: medium none ; border-collapse: collapse;" class="MsoTableGrid"><br />
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<td align="justify" width="614" valign="top" style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.6pt;"><br />
<p class="MsoNormal"><o:p><b>PLEDGE:</b> All experimental work on this project was performed from May to October 2007 by the undergraduate students participating in the team under the tutorial of instructors. All the students participated at iGEM for the first time.</o:p></p><br />
</td><br />
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</tbody><br />
</table><br />
<br />
<center><br />
<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td><a href="http://www.ki.si/">National Institute of Chemistry</a> <br><br />
<a href="http://www.ki.si/"><img border="0" src="https://static.igem.org/mediawiki/2007/2/29/KIlogo.gif" width="110" height="70"><br></a><br></td><br />
<td> </td><td> </td><td> </td><td> </td><br />
<br />
<td><br><a href="http://www.uni-lj.si/en">University of Ljubljana</a> <br><br />
<a href="http://www.uni-lj.si/en/"><img border="0" src="https://static.igem.org/mediawiki/2007/3/33/UL-logo.jpg"> <br></a></td><br />
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</tr><br />
</table><br />
</center><br />
<br />
<br />
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<br />
<br />
<div id="footer">______________________________________<br /><br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
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<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
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<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
<br />
<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<br><br><br />
</div> <br />
<br />
<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/User:RGaber/stylesheet/AC_RunActiveContent.jsUser:RGaber/stylesheet/AC RunActiveContent.js2007-11-23T18:23:29Z<p>RGaber: </p>
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case "ondeactivate":<br />
case "type":<br />
case "codebase":<br />
case "id":<br />
ret.objAttrs[args[i]] = args[i+1];<br />
break;<br />
case "width":<br />
case "height":<br />
case "align":<br />
case "vspace": <br />
case "hspace":<br />
case "class":<br />
case "title":<br />
case "accesskey":<br />
case "name":<br />
case "tabindex":<br />
ret.embedAttrs[args[i]] = ret.objAttrs[args[i]] = args[i+1];<br />
break;<br />
default:<br />
ret.embedAttrs[args[i]] = ret.params[args[i]] = args[i+1];<br />
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ret.objAttrs["classid"] = classid;<br />
if (mimeType) ret.embedAttrs["type"] = mimeType;<br />
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<div>#container {<br />
position: relative; <br />
margin: 0 auto;<br />
width: 932px;<br />
padding: 1px;<br />
text-align: left;<br />
background: #d0d2fb; <br />
}<br />
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/* intro */<br />
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#pageHeader {<br />
height: 190px;<br />
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background: url(https://static.igem.org/mediawiki/2007/7/70/Logo_brez_slike_modri.jpg) center top no-repeat;<br />
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clear: both;<br />
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#linkList {<br />
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#linkList a.two, #linkList a.two:link, #linkList a.two:visited {<br />
color: #FFFFFF; font-size: 110%; font-weight: bold;<br />
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#linkList a.two:hover {<br />
color: #FFF;<br />
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width: 150px; font-size:110%; font-weight: bold;<br />
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#linkList ul {<br />
list-style: none;<br />
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margin-top: 25px;<br />
margin-bottom: 10px;<br />
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background: #33356d;<br />
text-align: center;<br />
color: #FFFFFF;<br />
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<div>#container {<br />
position: relative; <br />
margin: 0 auto;<br />
width: 932px;<br />
padding: 1px;<br />
text-align: left;<br />
background: #d0d2fb; <br />
}<br />
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/* intro */<br />
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#pageHeader {<br />
height: 460px;<br />
margin: 0;<br />
padding: 0;<br />
background: url(https://static.igem.org/mediawiki/2007/1/10/Logo_s_sliko_modri.jpg) center top no-repeat;<br />
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}<br />
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#pageHeader h1 span {<br />
position: absolute;<br />
top: 68px;<br />
left: 44px;<br />
width: 250px; <br />
}<br />
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#pageHeader h2 span {<br />
position: absolute;<br />
top: 120px;<br />
left: 44px;<br />
width: 250px; <br />
}<br />
<br />
#quickSummary .p1 {<br />
position: absolute;<br />
top: 42px;<br />
left: 378px;<br />
width: 274px;<br />
font-size: 90%;<br />
height: 87px;<br />
}<br />
<br />
#quickSummary .p2 span {<br />
position: absolute;<br />
top: 138px;<br />
left: 378px;<br />
width: 275px; <br />
font-size: 80%;<br />
color: #000000;<br />
}<br />
<br />
#naslov {<br />
margin-left: 220px;<br />
margin-right: 35px;<br />
margin-bottom: 30px;<br />
padding: 10px 20px 10px;<br />
}<br />
<br />
#about {<br />
margin-left: 220px;<br />
margin-right: 35px;<br />
margin-bottom: 30px;<br />
padding: 10px 20px 10px;<br />
border-style: solid;<br />
border-width: 1px;<br />
border-color: #000000;<br />
}<br />
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#about h3 {<br />
color: #33356d;<br />
margin-bottom: 10px;<br />
font-weight: bold;<br />
}<br />
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margin-left: 220px;<br />
margin-right: 35px;<br />
margin-bottom: 30px;<br />
padding: 10px 20px 10px;<br />
border-style: solid;<br />
border-width: 1px;<br />
border-color: #000000;<br />
}<br />
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#aboutred h3 {<br />
color: #ed2224;<br />
margin-bottom: 10px;<br />
font-weight: bold;<br />
}<br />
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/* generic text */<br />
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#supportingText {<br />
margin-left: 200px;<br />
margin-right: 25px;<br />
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}<br />
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#products, #services, #support, #development {<br />
padding-right: 50px;<br />
}<br />
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#supportingText h3 {<br />
margin-bottom: 10px;<br />
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color: #000000;<br />
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clear: both;<br />
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margin-top: 60px;<br />
}<br />
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/* links */<br />
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font-size: 95%;<br />
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text-decoration: none; <br />
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}<br />
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#linkList a.two:hover {<br />
color: #FFF;<br />
text-decoration: none; <br />
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font-size: 100%;<br />
font-weight: bold;<br />
}<br />
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#llinks ul li {<br />
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}<br />
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#lresources {<br />
margin-bottom: 10px;<br />
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#lresults {<br />
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}<br />
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<div>Animation of split ubiquitin system</div>RGaberhttp://2007.igem.org/wiki/index.php/File:Split_tev.swfFile:Split tev.swf2007-11-23T18:01:52Z<p>RGaber: Animation of split TEV system</p>
<hr />
<div>Animation of split TEV system</div>RGaberhttp://2007.igem.org/wiki/index.php/File:Hiv_protease.swfFile:Hiv protease.swf2007-11-23T18:00:49Z<p>RGaber: Animation of HIV protease system</p>
<hr />
<div>Animation of HIV protease system</div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/methodsLjubljana/methods2007-10-27T00:17:54Z<p>RGaber: </p>
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<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
<link href="http://rokgaber.3host.biz/stylenew_zadaj.css" rel="stylesheet" type="text/css" /><br />
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<h3><span>Parts Design</span></h3><br />
<p class="p1"><span><br />
<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td> <img src ="https://static.igem.org/mediawiki/2007/d/da/Parts_design.JPG"> <br />
<small><b> Anja is preparing samples for plasmid isolation. </small></b></td><br />
<td> Since almost all of the parts used by our team had to be designed <i>de novo</i>, a huge portion of our time in the lab was spent on cloning. For each basic part, we designed primers containing standard BioBrick restriction sites. We had to introduce point mutations into some genes, because they contained one or more standard restriction sites inside the coding region. These mutations were introduced by PCR-driven overlap extension method (Heckman and Pease, 2007).<br> <br />
The genes, amplified by PCR, were therefore flanked by <i>Xba</i>I site upstream and <i>Spe</i>I, <i>Not</i>I and <i>Pst</i>I downstream of the gene. After restriction, fragments were cloned into <a href="http://partsregistry.org/Part:BBa_J52017">BBa_J52017</a>, a vector already containing a eukaryotic terminator sequence downstream of the cloning region. This vector has previously been constructed by the Slovenian iGEM 2006 team. </td><br />
</tr><br />
</table><br />
<br />
In addition to basic parts, we also designed dozens of composite parts, mainly constructs expressing chimeric proteins. Composite parts were assembled by three-point ligations, leaving 6 nucleotides long <i>Spe</i>I-<i>Xba</i>I mixed restriction site inbetween the two domains. Some of our composite parts contain as many as 6 basic parts. Such composite parts were constructed by repeating three point ligations.<br />
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</a></span></p><br><br />
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<div id="development"><br />
<h3><span>Cell cultures</span></h3><br />
<p class="p1"><br />
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<table border="0" style="background:#d0d2fb"><br />
<tr><br />
<td> In our experiments two different cell lines were used: human embrional kidney cell line HEK293T and human cervical cancer cell line HeLa. Cells were grown in DMEM medium containing 10% FBS and antibiotics streptomycin and penicillin, except during transfection when antibiotics were absent from the medium. For luciferase assays, cells were tipically seeded in 96-well plates, for microscopy in slide chambers, whereas for Western blotting and cytometry, where larger numbers of cells were needed, they were grown in 6-well plates, 35 mm dishes or even 10 cm<sup>2</sup> dishes. Depending on the type of experiment Lipofectamine 2000 or GeneJuice were used for transfection according to manufacturer´s protocol. Cells were transfected with the desired plasmids, stimulated if necessary and left grown before detection. </td><br />
<td> <img src ="https://static.igem.org/mediawiki/2007/5/53/Delo_s_kulturami.JPG"><small><b> Katja in cell lab - transfecting our cells. </small></b><br />
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<h3><span>Bioluminescence</span></h3><br />
<p class="p1">Bioluminescence is emission of light, produced by enzymatic reaction. In molecular biology, genes coding for enzymes, which catalize reactions where light is emitted, are used as reporter genes. The most widely used reporter enzymes are luciferases.<br><br />
<br />
In our experiments we used firefly luciferase placed under a specific inducible promoter of interest. The amount of sythesized luciferase was therefore dependent on the activation rate of cells and was semiquantified by measuring emitted light after substrate cleavage. As the number of cells and transfection efficiency vary, the results were normalized by a second reporter luciferase (the <i>Renilla</i> luciferase) placed under a constitutive promoter (expression of which is therefore cell activation independent) and with specificity for a different substrate. The measurements were conducted directly on 96-well plates in a luminometer.<br><br />
<br />
Luciferase assay proved to be our most valuable tool due to the extremely low detection limits. We used this assay to test both our split protein and HIV-protease systems. Cells were transfected with CD4 and CCR5 receptors fused with components of split ubiquitin or split TEV protease systems and reporter constructs. T7 promoter-luciferase reporter construct was used to measure the cellular response to viral attack. HIV surface protein g120 induces cleavage of of T7 RNA polymerase fused with a transmembrane segment and its translocation to nucleus, where it starts to transcribe the reporter gene. </p><br><br />
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<h3><span>Western blotting</span></h3><br />
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<td> <img src ="https://static.igem.org/mediawiki/2007/7/74/Blotanje.JPG"> <small><b> Andrej - specialist for Western blots. </small></b></td><br />
<td>Western blotting is a method of protein transfer from SDS-PAGE (SDS-polyacrylamide gel electrophoresis) gels to nitrocelulose membrane, which enables detection of proteins of interest in cell lysates using specific primary antibodies (pAb) and enzyme-conjugated secondary antibodies (sAb), which bind to pAb. In the first step, transfected cells grown in cell cultures were lysed and the insoluble membrane fraction was removed by centrifugation. Loading buffer containing SDS and a reducing agent was then added to the cleared lysate and the samples were denatured by boiling. Next, samples were applied to PAGE, where proteins were separated according to their molecular weight. Separated proteins were then transferred to a nitrocellulose membrane by electrical current. </td><br />
</tr><br />
</table><br />
<br />
The efficiency of the transfer was determined either by staining the gel with Ponceau dye or by using the pre-stained molecular weight markers, which are visible on the membrane after the transfer. After blotting, the free binding sites on the membrane were blocked by 5% non-fat dry milk in order to prevent unspecific binding of antibodies to the membrane. Then, the nitrocellulose membrane was incubated with pAb for 1 h at room temperature or overnight at 4<sup>o</sup>C with gentle shaking. The membrane was then washed to remove the unbound pAb and incubated with horseradish peroxidase-labelled sAb for 1 h at room temperature with gentle shaking. The signal was finally detected on the film by using a chemiluminescent substrate.<br><br />
Western blotting proved to be a very useful technique for determining whether proteolytic cleavage has occured. In the split ubiquitin assay, CMV-CD4-CUb-GFP and CMV-CCR5-NUb constructs were used. By comparing to molecular weight standards, we could detect whether the GFP was cut off from the transmembrane segment or remained attached to it when using anti-GFP antibodies. This method was also used in the HIV-protease project; the construct CMV-CD4-cleavage site-GFP was used and again GFPs of different sizes were detected when cotransfection with plasmid expressing HIV protease was performed.</p><br><br><br />
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<div id="development"><br />
<h3><span>Confocal microscopy</span></h3><br />
<p class="p1">The basis of fluorescence as a technique is excitation of a fluorophore, which then emits light of a specific wavelenght. The main feature of this technique is its very high sensitivity. Confocal microscopy enables three dimensional detection and allocation of flourescent molecules inside the cells. This technique was used to determine the location of CD4 fused to mCerulean or mCherry inside the cells. Additionally, microscopy of cells after transfection with CD4 and CCR5 receptor constructs fused with split protein systems or HIV-protease cleavage site was used to show that after cell activation (with gp120, pseudoviruses or HIV protease) cleavage of mCerulean or mCherry from the receptor occured. Thus, we can expect that the same occurs when T7 polimerase is fused instead of mCerulean or mCherry.<br> <br />
<br />
In our experiments, cell membranes were stained with SynaptoRed C2 (5 mM) for 5 minutes at 25 μM concentration and endoplasmic reticulums with ER-Tracker Red (1 mM) for 10 minutes at 2,5 μM. Cells were viewed using a confocal microscope with an oil-immersion objective. The 458, 514 (all multi-line Argon laser) and 543 (He-Ne laser) wavelengths were used to excite mCerulean, SynaptoRed C2 and mCherry (or ER-Tracker Red), respectively. </p><br><br />
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<div id="development"><br />
<h3><span>Flow cytometry</span></h3><br />
<p class="p1">Flow cytometry is a method which allows monitoring of cells in a flow stream on the basis of light scattering and fluorescence on single cell level. This method allows measuring of more parameters in a time and determination of a share of cells with a specific characteristic. There is a possibility of wide range of applications, however, we used two: annexin V and 7-AAD stainings for detection of apoptotic cells and antibody staining for detection of surface expression of transmembrane proteins.<br><br />
<br />
Apoptotic cells are characterized by certain morphological changes, one of them being translocation of phosphatidylserine (PS) from inner to the outer layer of plasma membrane bilayer. This change occurs early in the process. Annexin V is a protein, which binds specifically to PS. As this translocation of PS occurs early in the process, Annexin V is a sensitive probe to detect early apoptotic cells. Annexin V can be conjugated to a fluorochrome and thus detected by means of flow cytometry.<br> <br />
<br />
In addition to staining with Annexin V-PE (emission 575 nm), cells were also stained with 7-Amino actomycin (7-AAD, emission 670 nm), a dye that binds to necrotic cells. Staining of cells with both markers allows differentiation between early apoptotic cells, as they stain positive only for annexin, and necrotic cells, which stain positive for 7-AAD or both. This system was also used to present the idea as a whole - by using an effector protein which is an activator of apoptosis (such as caspase-3) as a reporter, binding of the virus to CD4 and CCR5 receptors expressed on cells would lead to their death even before the virus itself is able to replicate. This would then be reflected in positive staining of cells as detected by flow cytometry.<br><br />
<br />
Antibody staining was used for detection of transmembrane receptors CD4, CCR5 and CXCR4 on cell surfaces. For this purpose composite parts with receptors and Myc or HA tags, which flutter on the cell surface were designed. After transfection with plasmids expressing these receptors, cells were incubated with primary anti-tag antibodies, and then with secondary antibodies, which bind to constant, species-specific region of primary antibodies. Secondary antibodies were conjugated with a fluorophores (PE or DyeMer), which emit light at 575 nm and 610 nm, respectively. While using specific anti-Myc or anti-HA tag antibodies only cells transfected with our receptor constructs and not endogenous receptors were detected. </p><br><br />
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<div id="development"><br />
<h3><span>ELISA</span></h3><br />
<p class="p1">ELISA (enzyme-linked immunosorbent assay) is an immunological technique which enables semiquantitative determination of the amount of molecules of interest in the cell lysate using specific antibodies. Antibodies are bound to the surface of a microtiter plate and molecules of interest present in the cell lysate bind to them. The amount of bound molecules is detected using another primary antibody conjugated with biotin. Streptavidin conjugated with horse radish peroxidase specifically interacts with biotin. Peroxidase enzymatically cleaves a chromogenic substrate and the absorbance is measured. ELISA was used to determine the amount of caspase-3 protein after the activation of the engineered system with HIV protease, gp120 or pseudoviruses.</p><br><br />
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<div id="footer">______________________________________<br /><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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<h3><span>Performance of the Final Functional Systems</span></h3><br />
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<h3><span>Split systems based on heterodimerization</span></h3><br />
<p class="p1"><span><br />
After all subsystems were tested to be functional and their expression was optimized, final tests were performed to demonstrate that split-ubiquitin and split TEV protease systems were active. For the system to work there are some crucial steps:<br><br />
<ul><br />
<li>viral protein gp120 or (pseudo)viral particles have to bind to ectodomains of cell surface CD4 and CCR5<br />
<li>functional heterodimers of CD4 and CCR5 have to form<br />
<li>intracellular domains of CD4 and CCR5 have to bring split domains into close proximity <br />
<li>split domains have to reconstitute the functional protein (TEVP or ubiquitin)<br />
<li>T7 RNA polymerase needs to be cleaved off and translocated to the nucleus<br />
<li>T7 RNA polymerase has to start to transcribe the T7 promoter reporter gene<br />
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Our results demonstrate that split-ubiquitin and split TEVP systems were activated by the HIV coat protein gp120 which causes dimerization of CD4 and CCR5 receptors. Activation was amplified by the T7 RNAP system connected to luciferase reporter. Figures 4 and 5 show normalized luciferase activities of gp120-stimulated cells, nonstimulated and control cells, respectively in the split ubiquitin (Fig. 4) and split TEVP (Fig.5) system.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/2/2e/SASAslika1.jpg" width="633" height="380"><br><br />
<small> <b>Fig. 4. Viral envelope protein gp120 activates the split ubiquitin system.</b> Cells were transfected with CMV–CD4–CUb–T7RNA polymerase, CMV–CCR5–Nub, pT7-fLuc and CMV-rLuc reporter plasmids. 12 hours after transfection cells were activated with gp120 protein for additional 12 hours. Cells were lysed and luciferase activity was measured. The results were normalized for transfection efficiency and cell number using rLuc readings. Addition of gp120 clearly activated the split ubiquitin system as higher amounts of T7 promoter-regulated luciferase activity were measured. Luminescence was observed also in cells which were not stimulated, probably because of small level of spontaneusly dimerized CD4 and CCR5, as we used relatively strong promoter, which may cause high level receptors expression at the plasma membrane. T-test: p<0,01, ***; p<0,005, ****<br />
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<img src="https://static.igem.org/mediawiki/2007/8/86/Sasaslika2.jpg" width="633" height="353"><br><br />
<small><b>Fig. 5. Viral gp120 activates the split TEV protease system.</b> Cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal-TEVprotease site–T7 protease, pT7-fLuc and CMV-rLuc reporter plasmids. 12 hours after transfection cells were activated with gp120 protein for additional 12 hours. Cells were lysed and luciferase activity was measured. The results were normalized for transfection efficiency and cell number using rLuc readings. Addition of gp120 activated split TEV protease system as higher amounts of T7promoter luciferase were measured. Increased luminiscence was also observed in control cells, probably due to the same reason as in split ubiquitin experiment. T-test: p<0,1, *.T-test: p<0,005, ****.</small><br><br><br />
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<b>Interpretation of results:</b><br><br />
Firefly luciferase gene is regulated by the inducible T7 promoter, while renilla luciferase is under constitutive CMV promoter. The difference in luminescence of both luciferases is a consequence of different transfection efficiencies and cell number in different wells. Normalization means that we divide relative luminescence of firefly luciferase by relative luminescence of renilla luciferase. The ratio we get presents differences in promoter activity – the higher the ratio the more active promoter we have.<br><br><br />
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Measurements of luminescence revealed a higher firefly/renilla ratio in cells stimulated with viral protein gp120. A significant difference between stimulated and nonstimulated cells was observed. This is a strong evidence that the engineered device actually works as planned.<br><br><br />
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As seen in Figure 1, we could prove enhanced activation of T7 promoter in gp120 stimulated cells. To verify that the response is due only to T7 RNAP liberated from the ubiquitin fusion (and not e.g. by promoter leaking, spontaneous dimerization of CD4/CCR5, unspecific cleavage of membrane-attached T7 polymerase or unexpected activity of membrane-anchored T7 RNAP, we performed two additional control experiments.<br><br><br />
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Control 1: Cells were transfected with T7 promoter – firefly luciferase vector (and renilla luciferase for normalization standard). Only little background luminescence was detected. Control 2: Cells were transfected with both luciferase constructs and with CD4-CUb-T7 RNAP (control column in Figure 1). Luminescence was low, meaning that neither leaking of T7 promoter nor activity of T7 RNA polymerase on the plasma membrane (or its liberation from the bilayer) are the cause of T7 promoter activation.<br><br><br />
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Luminescence was observed also in non-stimulated cells. This could be due to spontaneous dimerization of CD4 and CCR5, probably because we used a relatively strong promoter which could lead to too many receptor molecules expressed and bound to the plasma membrane.<br><br><br />
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In the comparable experiment just with the split-TEVP attached to CD4 and CCR5 and T7 polymerase attached to the plasma membrane via a myristoyl group and connected to the receptor-split TEVP by a TEVP cleavage site. Results, presented in Figure 2 are highly comparable to those obtained with split ubiquitin, were controlled and interpreted as presented above for the split ubiquitin system.<br><br><br />
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In conclusion, with these experiments we proved that stimulation by HIV viral coat protein activated cellular response through both split-protein devices. We can expect that by replacing luciferase by an effector gene (caspase 3 or ß-interferon) cells would respond by activation of apoptosis or by synthesis of antiviral substances which would prevent the virus from spreading infection to healthy cells.<br><br><br />
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<b>Fluorescent confocal microscopy</b> was used to demonstrate the performance of the split TEV protease system and confirm luciferase data. Cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN and myristoylation signal–TEVprotease site–mCerulean. Fluorescence was detected at the plasma membrane, as expected (Fig. 6C). 22 hours after the transfection cells were stimulated with pseudovirus or gp120 protein and again observed 18 hours later. mCerulean was released from the membrane into cytosol in both cases (Fig. 6A and 6B). Additionally, membrane was stained after the stimulation to confirm the cytosolic localization, respectively (Fig. 6B).<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/b/b9/KATJA_slika6.jpg" width="633" height="217"><br><br />
<b><small>Fig. 6. HIV pseudovirus and gp120 protein activate split TEV protease system.</b> HEK293T cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal–TEVprotease site–mCerulean. 22 hours after transfection cells were stimulated with pseudovirus (A) or gp120 protein (B) for additional 18 hours. In both cases the split TEV protease system was activated and mCerulean reporter protein was released from membrane to cytosol. However, in unstimulated cells the system remained inactive and mCerulean was detected mainly at the plasma membrane (C).</small><br><br><br />
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We proposed that analogously T7 RNA polymerase fused to the receptor instead of mCerulean will be cleaved. Transfection with TEV protease cleavage site fused with T7 RNA polymerase with NLS and T7promoter–mCerulean was performed. Cells were stimulated with pseudovirus or gp120 protein 22 hours after transfection and again observed 18 hours later (Fig. 7). In stimulated cells intense expression of mCerulean was detected (Fig. 8A and 8B)whereas unstimulated cells remained clear (Fig. 7C). The system, using receptors CD4 and CCR5 with truncated cytoplasmic domains was also tested and resulted in similar results.<br><br><br />
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These experiments confirm that when activated, split TEV protease system releases T7 RNA polymerase from the membrane. Consequently T7 RNA polymerase translocates to the nucleus where it transcribes genes under the T7 promoter.<br><br><br />
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<img src="https://static.igem.org/mediawiki/2007/e/e1/KATJA_slika7.jpg" width="633" height="211"><br><br />
<b><small>Fig. 7. Activated split TEV protease system transcribes genes under the T7 promoter.</b> HEK293T cells were transfected with CMV–CD4–TEVC, CMV–CCR5–TEVN, myristoylation signal–TEVprotease site–T7 RNA polymerase-NLS and reporter plasmid T7-mCerulean. 22 hours after the transfection cells were stimulated with pseudovirus (A) or gp120 protein (B) for additional 18 hours. In both cases the split TEV protease system was activated, resulting in translocation of T7 RNA polymerase from the plasma membrane to the nucleus. Consequently mCerulean reporter protein was transcribed and detected in cytosol. In unstimulated cells the system remained inactive and no fluorescence at all was observed (C).</small><br><br />
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<h3><span>System based on the activation by HIV protease</span></h3><br />
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The process of system activation was observed using fluorescent confocal microscopy. Cells were transfected with CMV–CD4–HIV protease cleavage site–mCherry–NLS and optionally cotransfected with HIV protease on pNL4.3 plasmid. In cells without HIV protease fluorescence was detected at the plasma membrane (Fig. 8A), whereas in HIV protease cotransfected cells, fluorescent protein mCherry was released from the membrane and translocated to the nucleus (Fig. 8B). Similar results were obtained when mCherry was substituted with T7 RNA polymerase and additional construct pT7–mCerulean was transfected to detect the T7 RNA polymerase activity. In cells with HIV protease cotransfection, mCerulean was detected in cytosol (Fig. 9B), but no fluorescence at all was observed in the control experiment without of the HIV protease activity (Fig. 9A).<br><br><br />
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The system, using myristoylation signal instead of CD4 to anchor the T7 RNA polymerase to the membrane was also tested and we obtained similar results (data not shown).<br><br><br />
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We can conclude that in the presence of HIV protease, T7 RNA polymerase is released from the membrane and translocates to the nuceleus where it transcribes genes under the T7 promoter.<br><br><br />
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<a href="https://static.igem.org/mediawiki/2007/8/8d/KATJA_fig8.jpg"><br />
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<b><small>Fig. 8. HIV protease cleaves the linker between the membrane anchor and mCherry.</b> HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-mCherry-NLS (A) and additionally with HIV protease plasmid pNL4.3 (B). In cells without HIV protease, membrane localization of mCherry reporter protein was observed (A), but in the presence of HIV protease mCherry-NLS was released to cytosol and translocated to nucleus (B).</small><br><br><br />
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<b><small>Fig. 9. T7 RNA polymerase is activated by the cleavage of the linker on the membrane anchor by HIV protease.</b> HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-T7 RNA polymerase-NLS, reporter plasmid T7-mCerulean (A) and additionally with HIV protease plasmid pNL4.3 (B). HIV protease cut off T7 RNA polymerase from the membrane, thus activating it. Consequently mCerulean under the T7 promoter was transcribed and detected in cytosol (B). In the absence of protease the system remained inactive and no fluorescence was observed (A).</small><br />
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<h3><span>Effector protein – caspase-3</span></h3><br />
<p class="p1"><span><br />
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Instead of reporter proteins effector proteins such as caspase-3 were used, which are close to the real application. Caspase-3, a cysteine peptidase, which is the main executioner of the apoptosis. We have selected caspase-3 since we wanted to induce apoptosis in HIV-infected cells so that HIV could not replicate and infect the surrounding cells. Apoptosis prevents any inflammation to the neighboring cells and provides for a "clean removal" of apoptotic cells.<br><br><br />
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Two constructs were prepared for testing the caspase-3 activity: CMV-Cas3 and pT7-Cas3. The first construct was used to test if the caspase-3 is active in transfected cells. The second construct with caspase-3 under the T7 promoter was used as our main effector. Caspase-3 will be transcribed only when active T7 RNA polymerase is present in cytosol or nucleus. This should happen when our devices are activated by HIV virus-induced receptor heterodimerization or by HIV protease, expressed in HIV infected cells.<br><br><br />
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ELISA was used to determine the amount of caspase-3 in cells – this indicates the apoptotic potential of our system. We wanted to show that amount of caspase-3 in cells infected with HIV protease is greater than in non-infected cells (Fig. 10). The amount of caspase-3 had to be carefully monitored since the expression of caspase-3 leads to apoptosis and killing the cells, which we then can not detect anymore. So, timing of ELISA tests was therefore very important.<br><br><br />
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<a href="https://static.igem.org/mediawiki/2007/a/a4/CASPASE_fig10.jpg"><br />
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<b><small>Fig. 10. Caspase-3 expression is enchanced in HIV-protease transfected cells.</b> HeLa cells were transfected with effector gene pT7-Cas3 alone or with effector gene and components of system based on activation by HIV protease activity: CD4 membrane receptor with bound T7 RNA polymerase via HIV protease cleavage site and HIV protease in pNL4.3 vector. ELISA test was made with antibodies against human caspase-3. Significant amount of caspase-3 was detected in nontransfected cells and in cells, transfected with pT7-Cas3 as well. Since ELISA can also detect inactive and endogenous caspase-3 and since certain amount of cells is always in apoptosis, we did not expect extremely low values in these two samples. Significantly elevated levels of caspase-3 were detected in cells, transfected with our system components, meaning that more caspase-3 is transcribed and activated cells after HIV protease cuts off T7 RNA polymerase from transmembrane receptor. In terms of our initial idea this means that HIV entry and HIV protease activity in infected cells could trigger apoptosis and thus prevent replication of virus. T-test: p<0,05, **; p<0,01, ***.</small><br><br><br />
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Flow cytometry using annexin V staining was used for detection of amount of apoptotic cells as a consequence of caspase 3 activity after system activation. We have transfected cells with components of our systems (that have been previously proved as functional) with caspase-3 under T7 promoter as an effector gene. As a positive control we used cells, transfected with CMV-Cas3 construct. Activation of apoptosis varies between different cell lines and also the efficiency between different inducers varies significantly. Although some apoptosis was detected still an additional optimization is needed to obtain reliable results.<br><br><br />
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Instead of caspase-3, which triggers apoptosis in HIV infected cells we could use effector proteins that would act as antiviral agents. Good example is interferon-β. Interferons are very important in antiviral defense. They are usually secreted as a consequence of elevated levels of dsRNA in cells. Its effects include prevention of viral replication and stimulation of the immune response. In extreme cases it can also trigger apoptosis, probably trough increased production of p53 gene product.<br><br><br />
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We have not directly tested the activity of human interferon-β in HIV-infected cells but have deposited BioBricks containing interferon-β, pT7-interferon-β and CMV-interferon-β to the Registry.<br><br><br />
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<h3><span>Rok Gaber</span></h3><br />
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<img src="https://static.igem.org/mediawiki/2007/e/ea/RokGaberSEPIA.jpg" width="633" height="474"><br><br />
<b>Since a droplet of Ethidium Bromide gushed into my eyes and I tried to deactivate it with UV lamp, I have to wear sunglasses all the time.</b><br><br><br />
Last year of microbiology studies at Biotechnical Faculty (University in Ljubljana). Besides synthetic biology I am also interested in alternative fuels, microbic expression systems and other areas of industrial biotechnology.<br><br><br />
<b>Contact:</b><br><br />
rok.gaber(at)gmail.com <br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
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<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
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<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
<br />
<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<ul><br />
<br />
<br><br><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
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<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/Ljubljana" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/Ljubljana" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" /><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/implementationLjubljana/implementation2007-10-26T22:11:57Z<p>RGaber: </p>
<hr />
<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
<link href="http://rokgaber.3host.biz/stylenew_zadaj.css" rel="stylesheet" type="text/css" /><br />
</head><br />
<body><br />
<br />
<div id="container"><br />
<div id="intro"><br />
<div id="pageHeader"><br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="aboutred"><br />
<h3><span>Implementation</span></h3><br />
<p class="p1"><span><b></b></span></p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="supportingText"><br />
<br />
<br />
<br />
<br />
<div id="products"><br />
<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
<br> <br />
<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
<br />
<br />
<h3><span>Split ubiquitin system</span></h3><br />
<p class="p1"><br />
<br />
<br />
For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
<br />
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
<br />
T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
<br />
When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
<br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_ubiquitin.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_ubiquitin.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
Animation of split-ubiquitin system.<br><br />
<br />
<br><br />
</p> <br />
</div><br />
<br />
<br />
<br />
<div id="support"><br />
<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
<br />
For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
Animation of split-TEV protease system.<br><br><br />
<br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div id="development"><br />
<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
<br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/hiv_protease.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/hiv_protease.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
<br />
Animation of HIV protease system.<br><br />
<br />
<br />
</p><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<div id="footer">______________________________________<br /><br />
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<a href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a><br />
|<a href="https://2007.igem.org/Ljubljana">Home</a>|<br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="llinks"><br />
<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
</div><br />
<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
</div><br />
<br />
<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
<br><br><br />
</div> <br />
<br />
<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
</div><br />
<center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/Ljubljana" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/Ljubljana" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" /><br />
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</body><br />
</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/implementationLjubljana/implementation2007-10-26T22:09:37Z<p>RGaber: </p>
<hr />
<div><html><br />
<head><br />
<title>Company Name</title><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
<link href="http://rokgaber.3host.biz/stylenew_zadaj.css" rel="stylesheet" type="text/css" /><br />
</head><br />
<body><br />
<br />
<div id="container"><br />
<div id="intro"><br />
<div id="pageHeader"><br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="aboutred"><br />
<h3><span>Implementation</span></h3><br />
<p class="p1"><span><b></b></span></p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="supportingText"><br />
<br />
<br />
<br />
<br />
<div id="products"><br />
<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
<br> <br />
<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
<br />
<br />
<h3><span>Split ubiquitin system</span></h3><br />
<p class="p1"><br />
<br />
<br />
For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
<br />
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
<br />
T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
<br />
When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
<br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_ubiquitin.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_ubiquitin.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
<b>Animation</b> of split-ubiquitin system.<br><br />
<br />
<br><br />
</p> <br />
</div><br />
<br />
<br />
<br />
<div id="support"><br />
<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
<br />
For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
<b>Animation</b> of split-TEV protease system.<br><br><br />
<br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div id="development"><br />
<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
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<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/hiv_protease.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/hiv_protease.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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<b>Animation</b> of HIV protease system.<br><br />
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<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
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<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/implementationLjubljana/implementation2007-10-26T22:07:54Z<p>RGaber: </p>
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<h3><span>Implementation</span></h3><br />
<p class="p1"><span><b></b></span></p><br />
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<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
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<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
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<h3><span>Split ubiquitin system</span></h3><br />
<p class="p1"><br />
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For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
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It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
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T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
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When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
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<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_ubiquitin.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_ubiquitin.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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<b>Animation</b> of split-ubiquitin system.<br />
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<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
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For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
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<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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<b>Animation</b> of split-TEV protease system.<br><br />
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<div id="development"><br />
<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
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<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/hiv_protease.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/hiv_protease.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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<b>Animation</b> of HIV protease system.<br><br />
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<div id="footer">______________________________________<br /><br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
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<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
</ul><br />
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<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
</ul><br />
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<div id="lresults"><br />
<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
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</ul><br />
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<div id="ldiscussion"><br />
<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
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</div> <br />
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<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/LjubljanaLjubljana2007-10-26T22:02:24Z<p>RGaber: </p>
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<title>Company Name</title><br />
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<p class="p1"><span><b>Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.</b></span></p><br />
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<h3><span>Abstract for nonspecialists</span></h3><br />
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The main problem of effective treatment of HIV infection is rapid mutation rate which makes the virus resistant against drugs. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously. Our approach was to construct a defense system based on the principles of synthetic biology, which is based on viral function to activate the defense response in the attacked cell. The effect of mutations can thus be avoided since those mutations that cause the loss of the function also render the virus harmless. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on the viral maturation. Activation of any of them activates the antiviral cell defense or alternatively kills the infected cells, preventing further spread of infection. The same approach could be implemented for defense against other viral infections.<br />
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<h3><span>Scientific abstract</span></h3><br />
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We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. Two essential viral functions have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease.<br />
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Binding of virus to human cells causes formation of CD4-CCR5 heterodimers, which reconstitutes the split TEV protease. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of versatile effector genes, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same type function was successfully utilized in the implementation of the split ubiquitin system.<br />
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Our second approach was to exploit the viral protease, which cleaves the specific linker introduced between the T7 RNA polymerase and a cellular membrane anchor. Released T7 RNA polymerase subsequently activates the defense similar to that described with the split protein system. All three systems have been shown to work in human cells.<br />
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<b>Animation</b> of split-ubiquitin system.<br />
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<h3><span>Executive summary</span></h3><br />
<p class="p1"><span>HIV-1 virus is one of the most difficult targets for therapy because it hijacks our immune system and particularly because the virus mutates rapidly. This allows the selection of mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Currently the most effective therapy consists of an inhibitor cocktail that decreases the probability of HIV to overcome all target sequences at the same time.<br><br><br />
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We propose a different strategy, where we target a specific <b>FUNCTION</b> of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.<br><br><br />
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For implementation of this idea we have selected <b><u>two viral functions:</u></b> <b>viral attachment</b> to the cellular co-receptors CD4 and CCR5 and <b>viral proprotein processing</b> by its own protease.<br />
<br />
Binding of virus to T-cells leads to the formation of CD4-CCR5 heterodimer. In our device, formation of this heterodimer triggers reconstitution of a split protein (split ubiquitin or TEVP) which activates the specific proteolytic activity. This releases the T7 RNA polymerase from the membrane anchor and leads to transcription of the effector gene, which prevents the spread of viral infection.<br><br />
The second implementation of this idea was to utilize the HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 polymerase. Activation of viral protease similarly as above releases the T7 polymerase and starts the defense program.<br><br><br />
<br />
Important points of both approaches are to use the amplification of the signal, achieved by the use of T7 polymerase and the system's versatility, as we can select any number of different genes to be activated by the detection of viral function. The crucial aspect of our approach is that this system is <b>not sensitive to viral mutations</b> and is not activated only in case where the mutation leads to the loss of the viral function, which however also renders virus harmless at the same time.<br><br><br />
<br />
We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.<br><br><br />
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<p class="MsoNormal"><o:p><b>PLEDGE:</b> All experimental work on this project was performed from May to October 2007 by the undergraduate students participating in the team under the tutorial of instructors. All the students participated at iGEM for the first time.</o:p></p><br />
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<td><a href="http://www.ki.si/">National Institute of Chemistry</a> <br><br />
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<td><a href="http://www.uni-lj.si/en/">University of Ljubljana </a> <br><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/RokGaberLjubljana/RokGaber2007-10-26T22:00:01Z<p>RGaber: </p>
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<h3><span>Rok Gaber</span></h3><br />
<p class="p1"><span><br />
<br />
<img src="https://static.igem.org/mediawiki/2007/e/ea/RokGaberSEPIA.jpg" width="633" height="474"><br><br />
<b>Since a droplet of Ethydium Bromide gushed into my eyes and I tried to deactivate it (the idea of other team members) with UV lamp, I have to wear sunglasses all the time.</b><br><br><br />
I am in last year of undergraduate studies of microbiology at Biotechnical Faculty (University in Ljubljana). Besides in synthetic biology I am also interested in alternative fuels, microbic expression systems and other areas of industrial biotechnology.<br><br><br />
<b>Contact:<b><br><br />
rok.gaber(at)gmail.com <br />
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<p class="p1"><span><b>Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.</b></span></p><br />
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<h3><span>Abstract for nonspecialists</span></h3><br />
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The main problem of effective treatment of HIV infection is rapid mutation rate which makes the virus resistant against drugs. Current therapy uses combinations of different drugs, since it is less probable for the virus to develop the resistance against all of them simultaneously. Our approach was to construct a defense system based on the principles of synthetic biology, which is based on viral function to activate the defense response in the attacked cell. The effect of mutations can thus be avoided since those mutations that cause the loss of the function also render the virus harmless. We successfully implemented two types of defense devices – one based on the viral attachment to the cell and another based on the viral maturation. Activation of any of them activates the antiviral cell defense or alternatively kills the infected cells, preventing further spread of infection. The same approach could be implemented for defense against other viral infections.<br />
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<h3><span>Scientific abstract</span></h3><br />
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We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. Two essential viral functions have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease.<br />
<br />
Binding of virus to human cells causes formation of CD4-CCR5 heterodimers, which reconstitutes the split TEV protease. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of versatile effector genes, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same type function was successfully utilized in the implementation of the split ubiquitin system.<br />
<br />
Our second approach was to exploit the viral protease, which cleaves the specific linker introduced between the T7 RNA polymerase and a cellular membrane anchor. Released T7 RNA polymerase subsequently activates the defense similar to that described with the split protein system. All three systems have been shown to work in human cells.<br />
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<h3><span>Executive summary</span></h3><br />
<p class="p1"><span>HIV-1 virus is one of the most difficult targets for therapy because it hijacks our immune system and particularly because the virus mutates rapidly. This allows the selection of mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Currently the most effective therapy consists of an inhibitor cocktail that decreases the probability of HIV to overcome all target sequences at the same time.<br><br><br />
<br />
We propose a different strategy, where we target a specific <b>FUNCTION</b> of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.<br><br><br />
<br />
For implementation of this idea we have selected <b><u>two viral functions:</u></b> <b>viral attachment</b> to the cellular co-receptors CD4 and CCR5 and <b>viral proprotein processing</b> by its own protease.<br />
<br />
Binding of virus to T-cells leads to the formation of CD4-CCR5 heterodimer. In our device, formation of this heterodimer triggers reconstitution of a split protein (split ubiquitin or TEVP) which activates the specific proteolytic activity. This releases the T7 RNA polymerase from the membrane anchor and leads to transcription of the effector gene, which prevents the spread of viral infection.<br><br />
The second implementation of this idea was to utilize the HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 polymerase. Activation of viral protease similarly as above releases the T7 polymerase and starts the defense program.<br><br><br />
<br />
Important points of both approaches are to use the amplification of the signal, achieved by the use of T7 polymerase and the system's versatility, as we can select any number of different genes to be activated by the detection of viral function. The crucial aspect of our approach is that this system is <b>not sensitive to viral mutations</b> and is not activated only in case where the mutation leads to the loss of the viral function, which however also renders virus harmless at the same time.<br><br><br />
<br />
We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.<br><br><br />
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<td width="614" valign="top" style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.6pt;"><br />
<p class="MsoNormal"><o:p><b>PLEDGE:</b> All experimental work on this project was performed from May to October 2007 by the undergraduate students participating in the team under the tutorial of instructors. All the students participated at iGEM for the first time.</o:p></p><br />
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<td><a href="http://www.ki.si/">National Institute of Chemistry</a> <br><br />
<a href="http://www.ki.si/"><img border="0" src="https://static.igem.org/mediawiki/2007/2/29/KIlogo.gif" width="110" height="70"><br></a><br></td><br />
<td> </td><td> </td><td> </td><td> </td><br />
<br />
<td><a href="http://www.uni-lj.si/en/">University of Ljubljana </a> <br><br />
<a href="http://www.uni-lj.si/en/"><img border="0" src="https://static.igem.org/mediawiki/2007/e/ea/Menuleft_logo_unilj.gif" width="52" height="104"> <br></a> <br></td><br />
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<h3><span>Implementation</span></h3><br />
<p class="p1"><span><b></b></span></p><br />
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<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
<br> <br />
<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
<br />
<br />
<h3><span>Split ubiquitin system</span></h3><br />
<p class="p1"><br />
<br />
<br />
For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
<br />
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
<br />
T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
<br />
When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
<br />
<br />
<br />
<center><br />
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<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
<br />
For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
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<br />
<br />
<br />
<br />
<br />
<div id="development"><br />
<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
<br />
<br />
<br />
<center><br />
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<div id="footer">______________________________________<br /><br />
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<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
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<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
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<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
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<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
<ul><br />
<br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li><br />
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<h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3><br />
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</div> <br />
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<div id="ldiscussion"><br />
<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
</ul><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/implementationLjubljana/implementation2007-10-26T21:17:31Z<p>RGaber: </p>
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<h3><span>Implementation</span></h3><br />
<p class="p1"><span><b></b></span></p><br />
</div><br />
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<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
<br> <br />
<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
<br />
<br />
<h3><span>Split ubiquitin system</span></h3><br />
<p class="p1"><br />
<br />
<br />
For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
<br />
It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
<br />
T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
<br />
When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
<br />
<br />
<br />
<center><br />
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<div id="support"><br />
<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
<br />
For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
<br />
<br />
<center><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="600" height="500" id="team2" > <param name="allowScriptAccess" value="sameDomain" /> <param name="movie" value="http://rokgaber.3host.biz/split_tev.swf" /><param name="quality" value="high" /><param name="bgcolor" value="#ffffff" /><embed src="http://rokgaber.3host.biz/split_tev.swf" quality="high" bgcolor="#ffffff" width="600" height="500" name="team2" allowScriptAccess="sameDomain" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer" /><br />
</object><br><br />
</center><br />
<br />
<br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div id="development"><br />
<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
<br />
We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
<br />
When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
<br />
<br />
<br />
<center><br />
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</center><br />
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<br />
<h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li><br />
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<h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3><br />
<ul><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> <br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li><br />
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</div><br />
<div id="lresources"><br />
<h3 class="resources"><span><a class="two" href="https://2007.igem.org/Ljubljana/results">Results</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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<h2><span>Activation based on heterodimer formation and reconstitution of split proteins</span></h2><br />
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<p class="p1"><span>We selected to use dimerization of two human transmembrane receptors, CD4 and CCR5 (or CXCR4), as a signal for triggering the antiviral defense system. The advantage of the system is that antiviral processes in the cells start even before a virus infects cells. We discussed several possible approaches and finally focused on split proteins as a possible initiation point of a new signaling pathway. Two split proteins were found to be potentially useful (Stagljar and Fields, 2002; Wehr et al, 2006), ubiquitin and tobacco etch virus protease (TEVP), because they both result in a proteolytic event, which can liberate the next protein in the activation cascade.<br><br><br />
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For the purpose of our project we fused CD4 transmembrane receptor with the C-terminal part of ubiquitin (Cub), and CCR5 (or CXCR4) with the N-terminal part of ubiquitin (Nub). Our pathway could thus be induced by both HIV-1 and HIV-2 (which uses CXCR4 as the coreceptor for binding onto target cells). The basic idea behind our approach was that dimerization of receptors caused by HIV enables reconstitution of Nub and Cub. The reassembled ubiquitin is recognized by the ubiquitin-specific protease and cleaved at its C-terminus. If we append an effector protein onto the CD4-Cub fusion, the specific protease would thus release the effector, which is fixed on the membrane in an inactive form before dimerization. The principle of the split ubiquitin assay is described <a href="https://2007.igem.org/Ljubljana/splitubiquitinassay">HERE</a>.<br><br><br />
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It is likely that HIV causes dimerization of just a few CD4 and CCR5 receptors, therefore we could not rely on only a few effector protein molecules (e.g. caspase-3 or interferon β) being released. The release of just a few effector molecules would not be sufficient for a strong antiviral effect. We therefore decided to use the very specific bacteriophage T7 RNA polymerase (T7 RNAP), add a nuclear localization signal (NLS) to its end and couple both to the C-terminus of Cub.<br><br><br />
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T7 RNAP transcribes only genes controlled by the T7 bacteriophage promoter. This promoter is very strong and is not recognized by any other cellular RNA polymerase. In our devices either the death effector caspase 3 gene, which is part of the apoptosis pathway and causes controlled cell death, or interferon β gene, which has a role in the cellular antiviral defense system were placed under this promoter.<br><br><br />
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When HIV binds to the cell surface it triggers dimerization of CD4 and CCR5 (or CXCR4) receptors. In our synthetic system, receptors are linked to split ubiquitin and T7 RNAP-NLS. As a consequence of dimerization, T7 RNA polymerase is released from the membrane and translocates into the nucleus, where it transcribes the effector genes (could be one or several different!) under the control of the T7 promoter. This results either in apoptosis or in improved antiviral defense of the infected cell. We also designed a construct where T7 RNAP gene is placed under the T7 promoter for self amplification of the signal which enables mammalian cells to react to infection with minute numbers of HIV viruses.<br><br><br />
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<h3><span>Split TEV protease</span></h3><br />
<p class="p1">TEV protease (TEVP) is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease in molecular biotechnology is in removing affinity tags from purified recombinant proteins as it specifically cleaves the sequence ENLYFQ↓S. Additionally, a split protein assay was developed based on TEVP which functions very similar to the split ubiquitin system. The basic principle of this relatively new assay (Wehr et al, 2006) is described <a href="https://2007.igem.org/Ljubljana/splittevpassay">HERE</a>.<br><br><br />
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For the purpose of our project we fused CD4 receptor with TEVP-C (C-terminal half of TEV protease) and CCR5 co-receptor with TEVP-N (N-terminal half of TEV protease). Another fusion protein composed of the myristoylation domain, TEVP-specific cleavage site and T7 RNA polymerase with NLS was constructed. We named this protein ‘TEVP substrate protein’. T7 RNA polymerase was targeted to the membrane by connecting it to the membrane through a myristoyl anchor on the TEVP cleavage sequence. When released from the membrane, T7 RNAP translocates to the nucleus and transcribes genes controlled by the T7 promoter. Downstream of translocation of T7 RNA polymerase into the nucleus the system is the same as already described above under split ubiquitin system.<br><br><br />
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<h2><span>Activation based on the HIV Protease Activity</span></h2><br />
<p class="p1">The second approach to detect viral infection utilizes the proteolytic activity of HIV protease, so the system is useful when HIV has already infected cells. This protease is required for cleavage of a viral polyprotein and has a defined substrate specificity, similar to the above mentioned TEV protease. Activation of the viral protease inside infected cells can be used to trigger the defense system, as already tested with peptides (Vocero-Akbani, 1999). The activation step occurs when HIV protease cleaves inside the linker which connects the membrane anchor and a reporter protein.<br><br><br />
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We constructed two fusion proteins composed of a membrane anchor, linker with HIV proteolytic cleavage site and T7 RNA polymerase with NLS. The idea of the membrane anchor is to keep T7 RNAP connected to the membrane, so that in the absence of HIV virus the system remains inactive. In our case we used CD4 or myristoylation signal, but it could be essentially any other membrane protein.<br><br><br />
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When the expression of viral proteins for new virions starts, HIV protease becomes active in order to process viral proproteins. Active HIV protease can now also cleave and release T7 RNA polymerase from the membrane. T7 RNAP then translocates into the nucleus where it transcribes genes under the control of the T7 promoter as already described with previous systems.<br><br><br />
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<h3 class="results"><span><a class="two" href="https://2007.igem.org/Ljubljana/summary">Summary</a></span></h3><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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</html></div>RGaberhttp://2007.igem.org/wiki/index.php/Ljubljana/AndrejOndrackaLjubljana/AndrejOndracka2007-10-26T20:48:47Z<p>RGaber: </p>
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<h3><span>Andrej Ondračka</span></h3><br />
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At start of my studies I became interested in structural biology and biomolecular spectroscopy. Later, I also discovered the magical world of synthetic and systems biology. Currently, I am equally interested in both.<br><br />
Besides science, I like music, philosophy, photography, and my great passion, travelling.<br><br />
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For any information about me don't hesitate to contact me on andrej_ondracka (at) yahoo.com<br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li><br />
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<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li><br />
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li><br />
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