Ljubljana

From 2007.igem.org

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== Project in brief ==
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Last year our team modified human cells by introducing a feedback device with a dominant-negative inhibitor under the control of the NFkapaB promotor, which decreased the damaging excessive response to the bacterial infection. In 2007, we are remaining in the domain of the potential applications of synthetic biology to improve human health.
 
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The goal of our project is to design cells that will be able to detect the invasion of a pathogen and defend the organism by either undergoing apoptosis (and destroying the invaded pathogen before it can replicate) or by mounting any other defense response. To our opinion, the main innovative aspect of the project will be to detect the invasion of the pathogen not based on any particular sequence (which can be avoided through point mutations of the pathogen) but rather by relying on the ''function'' of the pathogen, which is essential for its survival or pathogenicity. The ultimate goal of this approach would be to develop gene therapy against infectious diseases.
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      <h3><span></span></h3>
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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 to assemble new cellular functions which are not yet existing in nature.</b></span></p>
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In parallel with the research, we prepared a 12-pages [http://2007.igem.org/Image:BrochureSB07_LJU_TitlePage.jpg brochure] on Synthetic Biology and our project in Slovenian language, aimed at life science students and general audience.
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      <h3><span>Abstract</span></h3>
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      <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 viral armies to select the mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Current therapy consists of an inhibitor cocktail that decreases the probability of HIV to modify all target sequences at the same time.<br><br>
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== Slovenian iGEM2007 team ==
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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>
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For implementation of this idea we have selected two functions: <b>viral attachment</b> to the cellular co-receptors CD4 and CCR5 and <b>viral proprotein processing</b> by its own protease.
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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>
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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>
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Ljubljana team in June 2007 (photo taken in the new meeting room at the [http://www.ki.si/index.php?id=117&no_cache=1&L=1 Natl. Institute of Chemistry]).<br>
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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>
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First row from left to right: Roman, Marko B., Sasa, Mojca, Karolina, Katja, Anja; second row from left to right: Peter, Andrej, Gabi, Mateja, Rok, Marko D. (missing on the photo is Nives)
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We have tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated on mammalian cell culture activation of response gene by infection with HIV-1 pseudovirus.<br><br>
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'''undergraduate students''':<br>
 
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Marko Bitenc - Biotechnology 3rd year<br>
 
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Peter Cimermančič - Biochemistry 3rd year<br>
 
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Rok Gaber - Microbiology 3rd year<br>
 
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Saša Jereb - Biochemistry 2nd year<br>
 
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Katja Kolar - Biochemistry 2nd year<br>
 
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Anja Korenčič - Biochemistry 4th year<br>
 
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Andrej Ondračka - Biochemistry 3rd year<br>
 
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'''instructors''':<br>
 
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Roman Jerala - NIC - ''supervisor''<br>
 
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Marko Dolinar - FCCT - ''supervisor''<br>
 
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Mojca Benčina - NIC<br>
 
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Karolina Ivičak - NIC<br>
 
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Mateja Manček Keber - NIC <br>
 
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Gabriela Panter - NIC<br>
 
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Nives Škrlj - FCCT<br>
 
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== Ljubljana and hosting institutions ==
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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>
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[http://www.ljubljana.si/en/ljubljana/ Ljubljana] (population 300.000) is the capital of [http://www.slovenia.si/ Slovenia] (population 2 million) and homes the largest of three universities in the country, [http://www.uni-lj.si University of Ljubljana] (UL). Most of the research work will be done at the [http://www.ki.si/index.php?id=117&no_cache=1&L=1 National Institute of Chemistry], [http://www.ki.si/index.php?id=192&no_cache=1&L=1 Laboratory of Biotechnology] and parts of it at the UL [http://www.fkkt.uni-lj.si/en/ Faculty of Chemistry and Chemical Technology], [http://openwetware.org/wiki/FCCT_Biochemistry_Lab Biochemistry Chair].
 
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== iGEM 2006 project ==
 
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Ljubljana team joined iGEM in 2006 and received the grand prize! The project focused on the signal transduction linked to development of sepsis. We engineered a negative feedback loop that inhibited excessive signalling via toll-like receptors. For more information, check the [http://2006.igem.org/wiki/index.php/Ljubljana%2C_Slovenia_2006 2006 wiki] or follow Alja's [http://partsregistry.org/movies/iGEM2006_ljubljana.mov presentation] at the Jamboree. We were also invited to [http://download.podcast.ethz.ch/media/MAVT-0001-00X/2007062409_dm.m4v present] our work at the 2007 SB 3.0 conference in Zurich.
 
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== ACKNOWLEDGEMENTS ==
 
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Our team would like to thank for the financial support to the organizations that made our research and attendance of the jamboree possible:
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    <div id="footer">______________________________________<br />
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AdFutura Public Fund,
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      |<a href="http://2007.igem.org/Ljubljana">Home</a>|
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Ministry of High Scool Education, Science and Technology of Slovenia,
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<a href="http://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a>
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University of Ljubljana,
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Slovenian Biochemical Society,
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European Commission - Synbiocomm Project,
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National Institute of Chemistry,
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Lek - a Sandoz company.
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[[Image:logo_glava_AdFutura.gif]]
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Special thanks are due to GeneArt for the support in synthesis of parts:
 
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[[Image:GeneArt.jpg]]
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        <h3 class="menu"><span><a class="two" href="http://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3>
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        <ul>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li>
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        <h3 class="links"><span><a class="two" href="http://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3>
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        <ul>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li>
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        <h3 class="resources"><span><a class="two" href="http://2007.igem.org/Ljubljana/results">Results</a></span></h3>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/subsystems">Subsystems Testing</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/finalsystem">Performance of the Final Functional Systems</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/biobricks">BioBricks</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/methods">Methods</a>&nbsp; </li>
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        <h3 class="results"><span><a class="two" href="http://2007.igem.org/Ljubljana/summary">Summary</a></span></h3>
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        <ul>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li>
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          <li><a class="one" href="http://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li>
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<div id="ldiscussion">
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        <h3 class="discussion"><span><a class="two" href="http://2007.igem.org/Ljubljana/team">Team</a></span></h3>
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Visitor location:<br>
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<li><a class="one" href="http://2007.igem.org/Ljubljana/glossary">Terms & References</a>&nbsp; </li>
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<li><a class="one" href="http://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li>
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Revision as of 19:23, 25 October 2007

Company Name

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 to assemble new cellular functions which are not yet existing in nature.

Abstract

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 viral armies to select the mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Current therapy consists of an inhibitor cocktail that decreases the probability of HIV to modify all target sequences at the same time.

We propose a different strategy, where we target a specific FUNCTION 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.

For implementation of this idea we have selected two functions: viral attachment to the cellular co-receptors CD4 and CCR5 and viral proprotein processing by its own protease. 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.
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.

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 not sensitive to viral mutations 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.

We have tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated on mammalian cell culture activation of response gene by infection with HIV-1 pseudovirus.

PLEDGE: 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.