http://2010.igem.org/wiki/index.php?title=Special:Contributions/Kristian&feed=atom&limit=50&target=Kristian&year=&month=2010.igem.org - User contributions [en]2024-03-28T20:12:51ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2011-01-14T15:23:11Z<p>Kristian: </p>
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<div class="virus"><img src="https://static.igem.org/mediawiki/2010/2/24/Freiburg10_rot250trans.gif" title="virus (3.5MB)" id="animated_virus"/></div><br />
<div class="div_home"><br />
<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
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<!---Box on the upper left: Project Results---><br />
<div class="box_home"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><img class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a>--><br />
Team Freiburg presents a functional, modular Virus Construction Kit for specifically targeting and killing tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold"><h5>Heading:</h5></span></a>--><br />
Structural engineering of the three dimensional viral capsid by introducing four distinct targeting sites led to successful differential transduction of tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a>--><br />
Efficient gene-directed enzyme prodrug activation was demonstrated by BioBrick-compatible constructs introduced into tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#arming"><span class="bold">[more]</span></a></p><br />
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<!---Big box on the left: Virus Construction Kit - Manual---><br />
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<div class="box_long"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a>--><br />
Build your own therapeutical virus – 150 pages of knowledge. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics" style="z-index:20;position:relative;"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p><br />
What happened in the lab during iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p>Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p>Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p>Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p>Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p>Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p>Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p>Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p>Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p>Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p>Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p>Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
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<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them. <a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><span class="bold">[more]</a></span></p><br />
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<!---Box on the lower left: Biosafety---><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">Aspects of biological safety were considered throughout and a risk assessment and security profile of the Adeno-associated Virus were created. Read more about Biosafety issues. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><span class="bold">[more]</span></a></p><br />
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<!---Box on the lower right: Notebook---><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold">From DNA to virus particle to cell culture? </span></a><br /><br />
Yes! Details on cloning, cell culture, flow cytometry analyses, quantifications by ELISA or Western Blot, cytotoxicity assays, time resolved microscopy ... <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold">[more]</span></a></p><br />
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<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><span class="bold">Fun and Science at the same time?</span></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest Cuckoo Clock Competition. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">[more]</span></a></p><br />
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<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.syntbio.net/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
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<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" 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://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a></center><br />
<br /><br />
<center><small>primary team contact: Kristian M. Müller; <a href="mailto:Kristian@syntbio.net">e-mail: Kristian@syntbio.net</a></small></center><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010Team:Freiburg Bioware/Team/Team 20102011-01-14T14:03:09Z<p>Kristian: </p>
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<h1>The iGEM 2010 Team Freiburg</h1><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/6/61/Freiburg10_Team_picture.png"<br />
style="width: 600px; height: auto; margin: 20px 0px 20px 170px;"><br />
<br />
<p style="text-align: justify;"><br />
We are a team<br />
of students<br />
consisting of 15 members. Among, there are 13 biology students<br />
specialized in<br />
various topics such as Biochemistry, Genetics, Microbiology, Plant<br />
Biotechnology, Immunology and of course Synthetic Biology. Furthermore,<br />
our<br />
team has one student of physics and one student of informatics.<br />
Combining these<br />
abilities we handled the challenges associated with our project and<br />
realized it<br />
hand in hand. Over the past summer, the team of biology students worked<br />
in<br />
groups and was greatly supported by our instructors and supervisor.<br />
Each group consisted<br />
of 2-3 students and was specialized either on DNA cloning or on cell<br />
culture.<br />
These main topics were again divided into smaller ones, like for<br />
example<br />
‘N-terminal fusion’ or ‘loop modifications’ of our virus. As our<br />
labjournal<br />
editings reached unlimited dimensions, we realized that we need further<br />
help. Luckily<br />
our Physics and Computer sciences students reinforced our team and<br />
designed the<br />
wiki and created mathematical models. <br><br />
Besides the<br />
daily<br />
laboratory work, we all spent some free time together, watching movies<br />
cooking,<br />
and having fun together. One of the funniest events was our canoe trip,<br />
sunny<br />
weather but however, wet conditions.<br><br />
For pictures see: [Link]</p><br />
<p style="text-align: justify;"><br><br />
</p><br />
<h1>Team members</h1><br />
<h2>Instructors</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/86/Freiburg10_Team_Kristian_Mueller.png"<br />
alt="Kristian Müller" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Kristian M. Müller</h5><br />
<p><br />
Kristian is our supervisor, he coordinates and reinforces us in our<br />
efforts and plans. <br><br />
You can reach him via his <a href="http://www.syntbio.net" target="_blank">web-site (http://www.syntbio.net)</a> or by <a href="mailto:Kristian@syntbio.net">e-mail (Kristian@syntbio.net)</a>.<br />
</p><br />
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</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Katja_Arndt.png"<br />
alt="Katja Arndt" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Katja M. Arndt</h5><br />
<p><br />
Katja was with us during the start-up phase but then had to leave us during the project, because she followed a call to a professorship in Potsdam. <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left"<br />
src="https://static.igem.org/mediawiki/2010/7/71/Freiburg10_Team_Sven_Hagen.png"<br />
alt="Sven Hagen" width="368" style="margin-right: 15px;"/><br />
<div><h5>Sven Hagen</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Tobi, of course. Thank you for everything.<br />
<br><br />
<br><br />
"Start by doing what's necessary; then do what's possible; and suddenly you are doing the impossible." (St. Francis of Assisi 1181/1182 – 1226)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><img class="left"<br />
src="https://static.igem.org/mediawiki/2010/3/31/Freiburg10_Team_Tobias_Baumann.png"<br />
alt="Tobias Baumann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Tobias Baumann</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Sven, of course. Thank you for everything.<br />
<br><br><br />
"If you're not part of the solution, you're part of the precipitate." (Henry J. Tillman)</p><br />
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<br />
<h2>Student Members</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/55/Freiburg10_Team_Achim_Mall_.png"<br />
alt="Achim Mall" width="368" style="margin-right: 15px;"/><br />
<div><h5>Achim Mall</h5><br />
<p><br />
Achim is surrounded by an aura of silence. Pipete more than 100 qPCRs?<br />
No problem! Keep cool and then let’s go.<br><br />
<br><br />
"I love deadlines. I like the whooshing sound they make as<br />
they fly by." (Douglas Adams)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_Team_Adrian_Fischer.png"<br />
alt="Adrian Fischer" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Adrian Fischer</h5><br />
<p><br />
This is Adrian, he is not only one of the best in cell culture, but he<br />
brought music from all centuries (preferring the 90’s!) into our lives.<br />
<br><br />
<br><br />
"You have to make the good out of the bad, because that is<br />
all you have got to make it out of." Robert Penn Warren<br />
</p><br />
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<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6d/Freiburg10_Team_Anissa_Bender.png"<br />
alt="Anissa Bender" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anissa Bender</h5><br />
<p><br />
Anissa has skills in cloning and cell culture works, both without any<br />
difficulty. And if you need somebody to be really honest, she is the<br />
right person.<br><br />
</p><br />
</div><br />
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<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_Team_Anna_Oschowitzer.png"<br />
alt="Anna Oschowitzer" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anna Oschowitzer</h5><br />
<p><br />
She`s the heart and soul of the lab. Always good-humored and poised to<br />
do that needs to be done. She has skills in cloning and PCR and did a<br />
good job in winning sponsors.<br />
<br><br><br />
"Research is what I'm doing when I don't know what I'm doing." (Wernher Von Braun)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_Team_Bea_Kaufmann.png"<br />
alt="Bea Kaufmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Bea Kaufmann</h5><br />
<p><br />
Our ‘laboratory smurf’ is an organizing talent and takes care of the<br />
whole project and its single members.<br />
<br><br />
<br><br />
"Good Morning!" (Unknown) --> Story to that: Leaving the lab at 4 a.m and being greeted from the security guard with a nice good morning! That is iGEM! Therefore, my citation of the month (or better: of the last months! ;-))<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/c/c3/Freiburg10_Team_Christian_Weing%C3%A4rtner.png"<br />
alt="Christian Weingärtner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Christian Weingärtner</h5><br />
<p><br />
He is the real midiprep master, the nanodrop is overstrained when<br />
measuring these enormous concentrations.<br />
<br><br />
<br><br />
"All the world's a stage" (William Shakespeare)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/7/7e/Freiburg10_Team_Hanna_Wagner.png"<br />
alt="Hanna Wagner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Hanna Wagner</h5><br />
<p><br />
The ITRs were nearly incomprehensible, until now! Here we have somebody<br />
with the fanciest methods and cloning strategies.<br/><br />
<br/><br />
"I believe there is no philosophical high-road in science, with epistemological signposts. No, we are in a jungle and find our way by trial and error, building our road behind us as we proceed."<br />
(Max Born) <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Team_Jessica_G%C3%BCnzle.png"<br />
alt="Jessica Günzle" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Jessica Günzle</h5><br />
<p><br />
Jessica is a multitalent. Planning our iGEM cajak tour, our trip and<br />
flight to Boston and working in the lab at the same time. That is<br />
great, man! <br><br />
<br><br />
"Equipped with his five senses, man explores the universe<br />
around him and calls the adventure Science." (Edwin Powell Hubble)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/84/Freiburg10_Team_Kerstin_Klingele.png"<br />
alt="Kerstin Klingele" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kerstin Klingele</h5><br />
<p><br />
Our sweet little girl does great work in cell culture and using flow<br />
cytometry. You can be sure with every business, it is all in good<br />
hands.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/d/d7/Freiburg10_Team_Kira_Meyerovich.png"<br />
alt="Kira Meyerovich" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kira Meyerovich</h5><br />
<p><br />
Laboratory experience is greatly available. And a very smart way of<br />
working, too.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_Team_Max_Schelker.png"<br />
alt="Max Schelker" width="368" style="margin-right: 15px;"/><br />
<div><h5>Max Schelker</h5><br />
<p><br />
Max is our physicist and a specialist in Systems Biology, he really<br />
knows how to handle data-based mathematical modeling. <br><br />
<br><br />
"Do not worry about your difficulties in Mathematics. I can<br />
assure you<br />
mine are still greater." (Albert Einstein)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Stefan_Bergmann.png"<br />
alt="Stefan Bergmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Stefan Bergmann</h5><br />
<p><br />
If you think there is no helping hand, you’re wrong. Stefan is<br />
everywhere and has got the plans in mind. <br><br />
<br><br />
"We are just an advanced breed of monkeys on a minor planet<br />
of a very average star. But we can understand the Universe. That makes<br />
us something very special."<br />
(S. Hawking)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/57/Freiburg10_Team_Volker_Morath.png"<br />
alt="Volker Morath" width="368" style="margin-right: 15px;"/><br />
<div><h5>Volker Morath</h5><br />
<p><br />
Guess who knows how to build a virus in his own basement. And if you<br />
need a straight opinion, go for Volker. <br><br />
<br><br />
"Quand tu veux construire un bateau, ne commence pas par<br />
rassembler du bois, couper des planches et distribuer du travail, mais<br />
reveille au sein des hommes le desir de la mer grande et large."<br />
(Antoine de Saint-Exupéry)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/4/4e/Freiburg10_Team_Patrick_Schindler.png"<br />
alt="Patrick Schindler" width="368" style="margin-right: 15px;"/><br />
<div><h5>Patrick Schindler</h5><br />
<p><br />
He is a hard worker, whether there is something to do in the<br />
laboratory, or to eat meals for a whole team. <br><br />
<br><br />
"If You Want Peace, prepare for war." (Publius Flavius Vegetius<br />
Renatus)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_Team_Timon.png"<br />
alt="Timon Schober" width="368" style="margin-right: 15px;"/><br />
<div><h5>Timon Schober</h5><br />
<p><br />
Do you see this great wiki? Yes, this is the work of our great web<br />
programmer and computer scientist Timon.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<h2>Friends</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Team_Fabian_Haas.png"<br />
alt="Fabian Haas" width="184" style="margin-right: 15px; margin-left:183px;"/><br />
<div><h5>Fabian Haas</h5><br />
<p><br />
Fabi helped us where he can: You're great!</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/be/Freiburg10_Team_Jo_Zang.png"<br />
alt="" width="368" style="margin-right: 15px;"/><br />
<div><h5>Johannes Zang</h5><br />
<p><br />
He takes the best pictures ever and knows how to fight with photoshop.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010Team:Freiburg Bioware/Team/Team 20102011-01-14T14:01:40Z<p>Kristian: </p>
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<html> <br />
<h1>The iGEM 2010 Team Freiburg</h1><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/6/61/Freiburg10_Team_picture.png"<br />
style="width: 600px; height: auto; margin: 20px 0px 20px 170px;"><br />
<br />
<p style="text-align: justify;"><br />
We are a team<br />
of students<br />
consisting of 15 members. Among, there are 13 biology students<br />
specialized in<br />
various topics such as Biochemistry, Genetics, Microbiology, Plant<br />
Biotechnology, Immunology and of course Synthetic Biology. Furthermore,<br />
our<br />
team has one student of physics and one student of informatics.<br />
Combining these<br />
abilities we handled the challenges associated with our project and<br />
realized it<br />
hand in hand. Over the past summer, the team of biology students worked<br />
in<br />
groups and was greatly supported by our instructors and supervisor.<br />
Each group consisted<br />
of 2-3 students and was specialized either on DNA cloning or on cell<br />
culture.<br />
These main topics were again divided into smaller ones, like for<br />
example<br />
‘N-terminal fusion’ or ‘loop modifications’ of our virus. As our<br />
labjournal<br />
editings reached unlimited dimensions, we realized that we need further<br />
help. Luckily<br />
our Physics and Computer sciences students reinforced our team and<br />
designed the<br />
wiki and created mathematical models. <br><br />
Besides the<br />
daily<br />
laboratory work, we all spent some free time together, watching movies<br />
cooking,<br />
and having fun together. One of the funniest events was our canoe trip,<br />
sunny<br />
weather but however, wet conditions.<br><br />
For pictures see: [Link]</p><br />
<p style="text-align: justify;"><br><br />
</p><br />
<h1>Team members</h1><br />
<h2>Instructors</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/86/Freiburg10_Team_Kristian_Mueller.png"<br />
alt="Kristian Müller" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Kristian M. Müller</h5><br />
<p><br />
Kristian is our supervisor, he coordinates and reinforces us in our<br />
efforts and plans. <br><br />
You can reach him via his <a href="http://www.syntbio.net" target="_blank">web-site (http://www.syntnbio.net)</a> or by <a href="mailto:Kristian@syntbio.net">e-mail (Kristian@syntbio.net)</a>.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Katja_Arndt.png"<br />
alt="Katja Arndt" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Katja M. Arndt</h5><br />
<p><br />
Katja was with us during the start-up phase but then had to leave us during the project, because she followed a call to a professorship in Potsdam. <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left"<br />
src="https://static.igem.org/mediawiki/2010/7/71/Freiburg10_Team_Sven_Hagen.png"<br />
alt="Sven Hagen" width="368" style="margin-right: 15px;"/><br />
<div><h5>Sven Hagen</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Tobi, of course. Thank you for everything.<br />
<br><br />
<br><br />
"Start by doing what's necessary; then do what's possible; and suddenly you are doing the impossible." (St. Francis of Assisi 1181/1182 – 1226)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><img class="left"<br />
src="https://static.igem.org/mediawiki/2010/3/31/Freiburg10_Team_Tobias_Baumann.png"<br />
alt="Tobias Baumann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Tobias Baumann</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Sven, of course. Thank you for everything.<br />
<br><br><br />
"If you're not part of the solution, you're part of the precipitate." (Henry J. Tillman)</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<br />
<h2>Student Members</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/55/Freiburg10_Team_Achim_Mall_.png"<br />
alt="Achim Mall" width="368" style="margin-right: 15px;"/><br />
<div><h5>Achim Mall</h5><br />
<p><br />
Achim is surrounded by an aura of silence. Pipete more than 100 qPCRs?<br />
No problem! Keep cool and then let’s go.<br><br />
<br><br />
"I love deadlines. I like the whooshing sound they make as<br />
they fly by." (Douglas Adams)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_Team_Adrian_Fischer.png"<br />
alt="Adrian Fischer" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Adrian Fischer</h5><br />
<p><br />
This is Adrian, he is not only one of the best in cell culture, but he<br />
brought music from all centuries (preferring the 90’s!) into our lives.<br />
<br><br />
<br><br />
"You have to make the good out of the bad, because that is<br />
all you have got to make it out of." Robert Penn Warren<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6d/Freiburg10_Team_Anissa_Bender.png"<br />
alt="Anissa Bender" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anissa Bender</h5><br />
<p><br />
Anissa has skills in cloning and cell culture works, both without any<br />
difficulty. And if you need somebody to be really honest, she is the<br />
right person.<br><br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_Team_Anna_Oschowitzer.png"<br />
alt="Anna Oschowitzer" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anna Oschowitzer</h5><br />
<p><br />
She`s the heart and soul of the lab. Always good-humored and poised to<br />
do that needs to be done. She has skills in cloning and PCR and did a<br />
good job in winning sponsors.<br />
<br><br><br />
"Research is what I'm doing when I don't know what I'm doing." (Wernher Von Braun)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_Team_Bea_Kaufmann.png"<br />
alt="Bea Kaufmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Bea Kaufmann</h5><br />
<p><br />
Our ‘laboratory smurf’ is an organizing talent and takes care of the<br />
whole project and its single members.<br />
<br><br />
<br><br />
"Good Morning!" (Unknown) --> Story to that: Leaving the lab at 4 a.m and being greeted from the security guard with a nice good morning! That is iGEM! Therefore, my citation of the month (or better: of the last months! ;-))<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/c/c3/Freiburg10_Team_Christian_Weing%C3%A4rtner.png"<br />
alt="Christian Weingärtner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Christian Weingärtner</h5><br />
<p><br />
He is the real midiprep master, the nanodrop is overstrained when<br />
measuring these enormous concentrations.<br />
<br><br />
<br><br />
"All the world's a stage" (William Shakespeare)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/7/7e/Freiburg10_Team_Hanna_Wagner.png"<br />
alt="Hanna Wagner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Hanna Wagner</h5><br />
<p><br />
The ITRs were nearly incomprehensible, until now! Here we have somebody<br />
with the fanciest methods and cloning strategies.<br/><br />
<br/><br />
"I believe there is no philosophical high-road in science, with epistemological signposts. No, we are in a jungle and find our way by trial and error, building our road behind us as we proceed."<br />
(Max Born) <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Team_Jessica_G%C3%BCnzle.png"<br />
alt="Jessica Günzle" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Jessica Günzle</h5><br />
<p><br />
Jessica is a multitalent. Planning our iGEM cajak tour, our trip and<br />
flight to Boston and working in the lab at the same time. That is<br />
great, man! <br><br />
<br><br />
"Equipped with his five senses, man explores the universe<br />
around him and calls the adventure Science." (Edwin Powell Hubble)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/84/Freiburg10_Team_Kerstin_Klingele.png"<br />
alt="Kerstin Klingele" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kerstin Klingele</h5><br />
<p><br />
Our sweet little girl does great work in cell culture and using flow<br />
cytometry. You can be sure with every business, it is all in good<br />
hands.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/d/d7/Freiburg10_Team_Kira_Meyerovich.png"<br />
alt="Kira Meyerovich" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kira Meyerovich</h5><br />
<p><br />
Laboratory experience is greatly available. And a very smart way of<br />
working, too.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_Team_Max_Schelker.png"<br />
alt="Max Schelker" width="368" style="margin-right: 15px;"/><br />
<div><h5>Max Schelker</h5><br />
<p><br />
Max is our physicist and a specialist in Systems Biology, he really<br />
knows how to handle data-based mathematical modeling. <br><br />
<br><br />
"Do not worry about your difficulties in Mathematics. I can<br />
assure you<br />
mine are still greater." (Albert Einstein)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Stefan_Bergmann.png"<br />
alt="Stefan Bergmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Stefan Bergmann</h5><br />
<p><br />
If you think there is no helping hand, you’re wrong. Stefan is<br />
everywhere and has got the plans in mind. <br><br />
<br><br />
"We are just an advanced breed of monkeys on a minor planet<br />
of a very average star. But we can understand the Universe. That makes<br />
us something very special."<br />
(S. Hawking)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/57/Freiburg10_Team_Volker_Morath.png"<br />
alt="Volker Morath" width="368" style="margin-right: 15px;"/><br />
<div><h5>Volker Morath</h5><br />
<p><br />
Guess who knows how to build a virus in his own basement. And if you<br />
need a straight opinion, go for Volker. <br><br />
<br><br />
"Quand tu veux construire un bateau, ne commence pas par<br />
rassembler du bois, couper des planches et distribuer du travail, mais<br />
reveille au sein des hommes le desir de la mer grande et large."<br />
(Antoine de Saint-Exupéry)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/4/4e/Freiburg10_Team_Patrick_Schindler.png"<br />
alt="Patrick Schindler" width="368" style="margin-right: 15px;"/><br />
<div><h5>Patrick Schindler</h5><br />
<p><br />
He is a hard worker, whether there is something to do in the<br />
laboratory, or to eat meals for a whole team. <br><br />
<br><br />
"If You Want Peace, prepare for war." (Publius Flavius Vegetius<br />
Renatus)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_Team_Timon.png"<br />
alt="Timon Schober" width="368" style="margin-right: 15px;"/><br />
<div><h5>Timon Schober</h5><br />
<p><br />
Do you see this great wiki? Yes, this is the work of our great web<br />
programmer and computer scientist Timon.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<h2>Friends</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Team_Fabian_Haas.png"<br />
alt="Fabian Haas" width="184" style="margin-right: 15px; margin-left:183px;"/><br />
<div><h5>Fabian Haas</h5><br />
<p><br />
Fabi helped us where he can: You're great!</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/be/Freiburg10_Team_Jo_Zang.png"<br />
alt="" width="368" style="margin-right: 15px;"/><br />
<div><h5>Johannes Zang</h5><br />
<p><br />
He takes the best pictures ever and knows how to fight with photoshop.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010Team:Freiburg Bioware/Team/Team 20102011-01-14T13:59:09Z<p>Kristian: </p>
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<h1>The iGEM 2010 Team Freiburg</h1><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/6/61/Freiburg10_Team_picture.png"<br />
style="width: 600px; height: auto; margin: 20px 0px 20px 170px;"><br />
<br />
<p style="text-align: justify;"><br />
We are a team<br />
of students<br />
consisting of 15 members. Among, there are 13 biology students<br />
specialized in<br />
various topics such as Biochemistry, Genetics, Microbiology, Plant<br />
Biotechnology, Immunology and of course Synthetic Biology. Furthermore,<br />
our<br />
team has one student of physics and one student of informatics.<br />
Combining these<br />
abilities we handled the challenges associated with our project and<br />
realized it<br />
hand in hand. Over the past summer, the team of biology students worked<br />
in<br />
groups and was greatly supported by our instructors and supervisor.<br />
Each group consisted<br />
of 2-3 students and was specialized either on DNA cloning or on cell<br />
culture.<br />
These main topics were again divided into smaller ones, like for<br />
example<br />
‘N-terminal fusion’ or ‘loop modifications’ of our virus. As our<br />
labjournal<br />
editings reached unlimited dimensions, we realized that we need further<br />
help. Luckily<br />
our Physics and Computer sciences students reinforced our team and<br />
designed the<br />
wiki and created mathematical models. <br><br />
Besides the<br />
daily<br />
laboratory work, we all spent some free time together, watching movies<br />
cooking,<br />
and having fun together. One of the funniest events was our canoe trip,<br />
sunny<br />
weather but however, wet conditions.<br><br />
For pictures see: [Link]</p><br />
<p style="text-align: justify;"><br><br />
</p><br />
<h1>Team members</h1><br />
<h2>Instructors</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/86/Freiburg10_Team_Kristian_Mueller.png"<br />
alt="Kristian Müller" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Kristian M. Müller</h5><br />
<p><br />
Kristian is our supervisor, he coordinates and reinforces us in our<br />
efforts and plans. <br><br />
You can reach him via his <a href="http://www.syntbio.net">web-site (http://www.syntnbio.net)</a> or by <a href="mailto:Kristian@syntbio.net">e-mail (Kristian@syntbio.net)</a>.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Katja_Arndt.png"<br />
alt="Katja Arndt" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Katja M. Arndt</h5><br />
<p><br />
Katja was with us during the start-up phase but then had to leave us during the project, because she followed a call to a professorship in Potsdam. <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left"<br />
src="https://static.igem.org/mediawiki/2010/7/71/Freiburg10_Team_Sven_Hagen.png"<br />
alt="Sven Hagen" width="368" style="margin-right: 15px;"/><br />
<div><h5>Sven Hagen</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Tobi, of course. Thank you for everything.<br />
<br><br />
<br><br />
"Start by doing what's necessary; then do what's possible; and suddenly you are doing the impossible." (St. Francis of Assisi 1181/1182 – 1226)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><img class="left"<br />
src="https://static.igem.org/mediawiki/2010/3/31/Freiburg10_Team_Tobias_Baumann.png"<br />
alt="Tobias Baumann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Tobias Baumann</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Sven, of course. Thank you for everything.<br />
<br><br><br />
"If you're not part of the solution, you're part of the precipitate." (Henry J. Tillman)</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<br />
<h2>Student Members</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/55/Freiburg10_Team_Achim_Mall_.png"<br />
alt="Achim Mall" width="368" style="margin-right: 15px;"/><br />
<div><h5>Achim Mall</h5><br />
<p><br />
Achim is surrounded by an aura of silence. Pipete more than 100 qPCRs?<br />
No problem! Keep cool and then let’s go.<br><br />
<br><br />
"I love deadlines. I like the whooshing sound they make as<br />
they fly by." (Douglas Adams)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_Team_Adrian_Fischer.png"<br />
alt="Adrian Fischer" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Adrian Fischer</h5><br />
<p><br />
This is Adrian, he is not only one of the best in cell culture, but he<br />
brought music from all centuries (preferring the 90’s!) into our lives.<br />
<br><br />
<br><br />
"You have to make the good out of the bad, because that is<br />
all you have got to make it out of." Robert Penn Warren<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6d/Freiburg10_Team_Anissa_Bender.png"<br />
alt="Anissa Bender" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anissa Bender</h5><br />
<p><br />
Anissa has skills in cloning and cell culture works, both without any<br />
difficulty. And if you need somebody to be really honest, she is the<br />
right person.<br><br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_Team_Anna_Oschowitzer.png"<br />
alt="Anna Oschowitzer" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anna Oschowitzer</h5><br />
<p><br />
She`s the heart and soul of the lab. Always good-humored and poised to<br />
do that needs to be done. She has skills in cloning and PCR and did a<br />
good job in winning sponsors.<br />
<br><br><br />
"Research is what I'm doing when I don't know what I'm doing." (Wernher Von Braun)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_Team_Bea_Kaufmann.png"<br />
alt="Bea Kaufmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Bea Kaufmann</h5><br />
<p><br />
Our ‘laboratory smurf’ is an organizing talent and takes care of the<br />
whole project and its single members.<br />
<br><br />
<br><br />
"Good Morning!" (Unknown) --> Story to that: Leaving the lab at 4 a.m and being greeted from the security guard with a nice good morning! That is iGEM! Therefore, my citation of the month (or better: of the last months! ;-))<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/c/c3/Freiburg10_Team_Christian_Weing%C3%A4rtner.png"<br />
alt="Christian Weingärtner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Christian Weingärtner</h5><br />
<p><br />
He is the real midiprep master, the nanodrop is overstrained when<br />
measuring these enormous concentrations.<br />
<br><br />
<br><br />
"All the world's a stage" (William Shakespeare)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/7/7e/Freiburg10_Team_Hanna_Wagner.png"<br />
alt="Hanna Wagner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Hanna Wagner</h5><br />
<p><br />
The ITRs were nearly incomprehensible, until now! Here we have somebody<br />
with the fanciest methods and cloning strategies.<br/><br />
<br/><br />
"I believe there is no philosophical high-road in science, with epistemological signposts. No, we are in a jungle and find our way by trial and error, building our road behind us as we proceed."<br />
(Max Born) <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Team_Jessica_G%C3%BCnzle.png"<br />
alt="Jessica Günzle" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Jessica Günzle</h5><br />
<p><br />
Jessica is a multitalent. Planning our iGEM cajak tour, our trip and<br />
flight to Boston and working in the lab at the same time. That is<br />
great, man! <br><br />
<br><br />
"Equipped with his five senses, man explores the universe<br />
around him and calls the adventure Science." (Edwin Powell Hubble)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/84/Freiburg10_Team_Kerstin_Klingele.png"<br />
alt="Kerstin Klingele" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kerstin Klingele</h5><br />
<p><br />
Our sweet little girl does great work in cell culture and using flow<br />
cytometry. You can be sure with every business, it is all in good<br />
hands.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/d/d7/Freiburg10_Team_Kira_Meyerovich.png"<br />
alt="Kira Meyerovich" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kira Meyerovich</h5><br />
<p><br />
Laboratory experience is greatly available. And a very smart way of<br />
working, too.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_Team_Max_Schelker.png"<br />
alt="Max Schelker" width="368" style="margin-right: 15px;"/><br />
<div><h5>Max Schelker</h5><br />
<p><br />
Max is our physicist and a specialist in Systems Biology, he really<br />
knows how to handle data-based mathematical modeling. <br><br />
<br><br />
"Do not worry about your difficulties in Mathematics. I can<br />
assure you<br />
mine are still greater." (Albert Einstein)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Stefan_Bergmann.png"<br />
alt="Stefan Bergmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Stefan Bergmann</h5><br />
<p><br />
If you think there is no helping hand, you’re wrong. Stefan is<br />
everywhere and has got the plans in mind. <br><br />
<br><br />
"We are just an advanced breed of monkeys on a minor planet<br />
of a very average star. But we can understand the Universe. That makes<br />
us something very special."<br />
(S. Hawking)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/57/Freiburg10_Team_Volker_Morath.png"<br />
alt="Volker Morath" width="368" style="margin-right: 15px;"/><br />
<div><h5>Volker Morath</h5><br />
<p><br />
Guess who knows how to build a virus in his own basement. And if you<br />
need a straight opinion, go for Volker. <br><br />
<br><br />
"Quand tu veux construire un bateau, ne commence pas par<br />
rassembler du bois, couper des planches et distribuer du travail, mais<br />
reveille au sein des hommes le desir de la mer grande et large."<br />
(Antoine de Saint-Exupéry)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/4/4e/Freiburg10_Team_Patrick_Schindler.png"<br />
alt="Patrick Schindler" width="368" style="margin-right: 15px;"/><br />
<div><h5>Patrick Schindler</h5><br />
<p><br />
He is a hard worker, whether there is something to do in the<br />
laboratory, or to eat meals for a whole team. <br><br />
<br><br />
"If You Want Peace, prepare for war." (Publius Flavius Vegetius<br />
Renatus)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_Team_Timon.png"<br />
alt="Timon Schober" width="368" style="margin-right: 15px;"/><br />
<div><h5>Timon Schober</h5><br />
<p><br />
Do you see this great wiki? Yes, this is the work of our great web<br />
programmer and computer scientist Timon.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<h2>Friends</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Team_Fabian_Haas.png"<br />
alt="Fabian Haas" width="184" style="margin-right: 15px; margin-left:183px;"/><br />
<div><h5>Fabian Haas</h5><br />
<p><br />
Fabi helped us where he can: You're great!</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/be/Freiburg10_Team_Jo_Zang.png"<br />
alt="" width="368" style="margin-right: 15px;"/><br />
<div><h5>Johannes Zang</h5><br />
<p><br />
He takes the best pictures ever and knows how to fight with photoshop.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Project/ResultsTeam:Freiburg Bioware/Project/Results2011-01-14T13:36:54Z<p>Kristian: </p>
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<h1>Summary</h1><br />
<p><br />
Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.<br />
</p><br />
<p><br />
During the development of our system, we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows for the exchange of two loops of the virus for either a His-tag, which enables purification, or a biotinylation-tag, or an antibody binding module. We demonstrated the functional assembly of viruses with all three modifications.<br />
</p><br />
<p><br />
In addition, we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.<br />
</p><br />
<p><br />
We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.<br />
</p><br />
<p><br />
Last but not least, we provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.<br />
</p><br />
<br />
<br><br />
Our key achievements are:<br />
<ul><br />
<li>A BioBrick set and assembled plasmids for gene delivery.<br><br />
This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.</li><br />
<li>A Biobrick set and plasmids for viral capsid production and modification.<br><br />
This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.</li><br />
<li>An extensive set of experiments demonstrating the functionality of our constructs.</li><br />
</ul><br />
</p><br />
<br /><br />
<br /><br />
<br />
<a name="highlights" class="onlyAnchor"><h2>Highlights</h2></a><br />
<br />
<br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid" name="modularization" class="onlyAnchor"><h2>Modularization</h2></a><br />
<p><br />
We converted a recombinant Adeno-associated virus to the BioBrick standard. This modularization is grouped into two parts: first, the generation of a gene of interest (vector) plasmid and second, the generation of vectors for the modified capsid proteins.<br />
</p><br />
<p><br />
<b><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Gene of Interest (GOI) Plasmid</a></b><br /><br />
The Virus Construction Kit enables researchers to encapsidate virtually any given DNA sequence into AAV-2 particles. As one example from therapeutic focus of our project, prodrug-activating enzymes are provided within the kit for efficient tumor cell killing. Additionally included fluorescent proteins allow monitoring of transduced cells by fluorescence microscopy and flow cytometry. Transgene expression can be fine-tuned using promoters of different specificity and enhancer elements also provided. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a><br />
</p><br />
<p><br />
<b><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Capsid Plasmids</a></b><br /><br />
AAV-2 genes essential for the production of viral particles in a specialized cell line were identified by literature search, isolated on genetic level and modified to meet the requirements of BioBrick assembly. For this purpose, a specialized variant of the iGEM default backbone was created, proven functional and submitted. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a><br /><br />
</p><br />
</td><br />
<td><br />
<table class="highlights"><br />
<tr><br />
<td><br />
<img width="115px" src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png" alt="banner"/><br />
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<td><br />
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</td><br />
</tr><br />
<tr><br />
<td><br />
<img width="115px" src="https://static.igem.org/mediawiki/2010/8/83/Freiburg10_FACS_withbetaglobin.png" alt="banner"/><br />
</td><br />
<td><br />
<img width="115px" src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png" alt="banner"/><br />
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</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
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<td><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion" name="targeting" class="onlyAnchor"><h2>Targeting</h2></a><br />
<p>Retargeting of the viral particles was achieved by knocking down the natural tropism of the Adeno-associates virus particles and at the same time introducing capsid modifications for specifically targeting tumor cells. For this purpose, two different strategies were developed, which were <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/insertion of motifs into surface-exposed loops">targeting via loops</a> or <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">targeting via fusion to the N-terminus of the viral protein VP2</a>.<br><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming" name="arming" class="onlyAnchor"><h2>Arming</h2></a><br />
<p>The specifically targeted tumor cells were killed by prodrug activation approaches. Viral particles were charged with thymidine kinase or cytosine deaminase constructs to kill cancer cells upon delivery of ganciclovir or 5-Fluorocytosine, respectively. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming">[more]</a><br />
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<a name="superconstruct" class="onlyAnchor"><h2>Tumor Killing</h2></a><br />
<p><br />
Finally, we successfully combined the various modifications of the viral vectors and demonstrated the use for tumor therapy applications. These modifications include the knock down of the natural tropism for HSPG receptor, the tumor targeting using the affibody ZEGFR1907 fused to the N-terminus of the viral coat protein VP2 and an encapsidated vector genome that was bricked and reassembled containing the prodrug converting Guanosine Monophosphate Kinase Thymidine Kinase fusion protein (mGMK-TK). Upon addition of ganciclovir only A431 tumor cells were killed but not the HeLa control cells.<br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Modeling" name="modeling" class="onlyAnchor"><h2>Modeling</h2></a><br />
<p><br />
Efforts in the lab were accompanied by mathematical modeling of the viral production and infection and the attempt to fit the models to time-lapse microscopy data. Planning of fusion proteins and loop modifications was based on structural analysis and modeling of the viral capsid, based on known crystal structrues. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Modeling" name="modeling">[more]</a><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Project/ResultsTeam:Freiburg Bioware/Project/Results2011-01-14T13:27:15Z<p>Kristian: </p>
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<h1>Summary</h1><br />
<p><br />
Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.<br />
</p><br />
<p><br />
During the development of our system, we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows for the exchange of two loops of the virus for either a His-tag, which enables purification, or a biotinylation-tag, or an antibody binding module. We demonstrated the functional assembly of viruses with all three modifications.<br />
</p><br />
<p><br />
In addition, we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.<br />
</p><br />
<p><br />
We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.<br />
</p><br />
<p><br />
Last but not least, we provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.<br />
</p><br />
<br />
<br><br />
Our key achievements are:<br />
<ul><br />
<li>A BioBrick set and assembled plasmids for gene delivery.<br><br />
This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.</li><br />
<li>A Biobrick set and plasmids for viral capsid production and modification.<br><br />
This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.</li><br />
<li>An extensive set of experiments demonstrating the functionality of our constructs.</li><br />
</ul><br />
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<a name="highlights" class="onlyAnchor"><h2>Highlights</h2></a><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid" name="modularization" class="onlyAnchor"><h2>Modularization</h2></a><br />
<p><br />
We converted a recombinant Adeno-associated virus to the BioBrick standard. This modularization is grouped into two parts: first, the generation of a gene of interest (vector) plasmid and second, the generation of vectors for the modified capsid proteins.<br />
</p><br />
<p><br />
<b><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Gene of Interest (GOI) Plasmid</a></b><br /><br />
The Virus Construction Kit enables researchers to encapsidate virtually any given DNA sequence into AAV-2 particles. As one example from therapeutic focus of our project, prodrug-activating enzymes are provided within the kit for efficient tumor cell killing. Additionally included fluorescent proteins allow monitoring of transduced cells by fluorescence microscopy and flow cytometry. Transgene expression can be fine-tuned using promoters of different specificity and enhancer elements also provided. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a><br />
</p><br />
<p><br />
<b><a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Capsid Plasmids</a></b><br /><br />
AAV-2 genes essential for the production of viral particles in a specialized cell line were identified by literature search, isolated on genetic level and modified to meet the requirements of BioBrick assembly. For this purpose, a specialized variant of the iGEM default backbone was created, proven functional and submitted. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a><br /><br />
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<img width="115px" src="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Overview_BBa_Vectorplasmid.png" alt="banner"/><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion" name="targeting" class="onlyAnchor"><h2>Targeting</h2></a><br />
<p>Retargeting of the viral particles was achieved by knocking down the natural tropism of the Adeno-associates virus particles and at the same time introducing capsid modifications for specifically targeting tumor cells. For this purpose, two different strategies were developed, which were <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/insertion of motifs into surface-exposed loops">targeting via loops</a> or <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">targeting via fusion to the N-terminus of the viral protein VP2</a>.<br><br />
</p><br />
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<table class="highlights"><br />
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<img width="115px" src="https://static.igem.org/mediawiki/2010/5/54/Freiburg_Targeting_Fig11c.png" alt="banner"/><br />
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<img width="115px" src="https://static.igem.org/mediawiki/2010/2/2e/Freiburg10_Loopover1.jpeg" alt="banner"/><br />
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<img width="115px" src="https://static.igem.org/mediawiki/2010/7/7a/Freiburg10_Loopover2.jpeg" alt="banner"/><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming" name="arming" class="onlyAnchor"><h2>Arming</h2></a><br />
<p>The specifically targeted tumor cells were killed by prodrug activation approaches. Viral particles were charged with thymidine kinase or cytosine deaminase constructs to kill cancer cells upon delivery of ganciclovir or 5-Fluorocytosine, respectively. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming">[more]</a><br />
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<a name="superconstruct" class="onlyAnchor"><h2>Tumor Killing</h2></a><br />
<p><br />
Finally we successfully combined different modifactions of the therapeutic viral vector for optimizing it for tumore therapy application.<br />
These modifications include the knock down of the natural tropism for HSPG receptor, the tumor targeting using the affibody ZEGFR1907 fused to the N-terminus of the viral coat protein VP2 and an encapsidated vectorgenome that was bricked down and reassembled containing the prodrug converting Guanosine Monophosphate Kinase Thymidine Kinase fusion protein (mGMK-TK).<br />
</p><br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Modeling" name="modeling" class="onlyAnchor"><h2>Modeling</h2></a><br />
<p><br />
Efforts in the lab were accompanied by mathematical modeling of the viral production and infection and the attempt to fit the models to time-lapse microscopy data. Planning of fusion proteins and loop modifications was based on structural analysis and modeling of the viral capsid, based on known crystal structrues. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Modeling" name="modeling">[more]</a><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/BioBricksTeam:Freiburg Bioware/BioBricks2011-01-14T12:21:45Z<p>Kristian: </p>
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<h1>Freiburg Bioware's 2010 iGEM Project BioBrick Parts</h1><br />
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In the list below you find an overview over the BioBrick parts, which were created by the iGEM team Freiburg_Bioware 2010 and which are provided within the Virus Construction Kit. This kit comprises various parts that can be used to produce therapeutic viral vectors based on the Adeno-associated Virus (AAV-2). For clarity and better usability we sorted the parts into categories that can also be found in the parts registry:<br />
<ul><br />
<li>//viral_vectors</li> <br />
<li>//viral_vectors/aav</li><br />
<li>//viral_vectors/aav/capsid_coding</li> <br />
<li>//viral_vectors/aav/vector_plasmid</li><br />
<li>//viral_vectors/aav/vector_miscellaneous</li><br><br />
</ul><br />
For the production of different therapeutic viral particles three types of plasmid are transfected: (i) one to two capsid coding plasmids that code for (i.i) the wild-type part of the capsid proteins and the rep proteins and (i.ii) a modified capsid protein for retargeting, (ii) a vector plasmid that contains the ITRs and is armed with a therapeutic gene of interest (GOI) that will be packaged into the viral capsid, and (iii) the helper plasmid. The third category of parts contains also miscellaneous plasmids, which are related or required for the Virus Construction Kit.<br />
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<h1>The Virus Construction Kit categories - click to view</h1><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks/capsid_coding"><img src="http://partsregistry.org/wiki/images/2/25/Freiburg10_Ue_Capsid_coding_plasmid.png" alt="Capsid coding parts" width="270px" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks/vector_plasmid" ><img src="http://partsregistry.org/wiki/images/2/2c/Freiburg10_Ue_Vector_plasmid.png" alt="Vector plasmid" width="270px" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks/miscellaneous" ><img src="http://partsregistry.org/wiki/images/2/2c/Freiburg10_Ue_Vector_plasmid.png" alt="Miscellaneous parts" width="270px" /></a><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2011-01-14T11:56:54Z<p>Kristian: </p>
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<p><br />
Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.<br />
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<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results"><h2>Project Results</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><h5>Highlights: </h5></a>--><br />
Team Freiburg presents a functional, modular Virus Construction Kit for specifically targeting and killing tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#highlights"><span class="bold"><h5>Heading:</h5></span></a>--><br />
Structural engineering of the three dimensional viral capsid by introducing four distinct targeting sites led to successful differential transduction of tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting"><span class="bold">[more]</span></a></p><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold"><h5>Head-Line:</h5></span></a>--><br />
Efficient gene-directed enzyme prodrug activation was demonstrated by BioBrick-compatible constructs introduced into tumor cells. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#arming"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!---Big box on the left: Virus Construction Kit - Manual---><br />
<br />
<div class="box_long"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Manual_Logo_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h2>Virus Construction Kit - The Manual</h2></a><br />
<p><!--<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><h5>Heading:</h5></a>--><br />
Build your own therapeutical virus – 150 pages of knowledge. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Virus_Construction_Kit"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!--- News Ticker ---><br />
<br />
<div class="box_long box_long_news news_ticker"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics" style="z-index:20;position:relative;"><h2>Statistics - In a Minute</h2></a><br />
<ul id="news" class="newsticker"><br />
<li><div class="news"><img class="right" src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Number_of_Plasmids.png" id="ccc" /><p><br />
What happened in the lab during iGEM? </p><br />
</div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_Longest_Wikipage.png"<br />
alt="Longest Wikipage" width="368"><p>Longest Wikipage:<br />with 222,795 bytes</p></div><br />
</li><br />
<li><div class="news"><img src="https://static.igem.org/mediawiki/2010/1/10/Freiburg10_Number_of_Biobricks.png"<br />
alt="Number of BioBricks" width="368"><p>Number of Biobricks:<br /> 116</p></div></li><br />
<li><div class="news"><img heigth="100px" src="https://static.igem.org/mediawiki/2010/3/3f/Freiburg10_Number_of_ordered_Oligo-Nucleotides.png"<br />
alt="Number of ordered Oligo-Nucleotides" width="184"><p>Number of Ordered Oligos:<br /> 193</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f3/Freiburg10_Minipreps_total.png"<br />
alt="Minipreps total" width="184"><p>Mini-Preps total:<br /> 1085</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_Midipreps_total.png"<br />
alt="Midipreps total" width="368"><p>Midi-Preps total:<br /> 106</p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d6/Freiburg10_Highest_Prep_concentration.png"<br />
alt="Highest Prep concentration (Midi)" width="368"><p>Highest Midi-Prep Concentration:<br />5045,6 ng/µL </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Number_of_Glyzerolstocks.png"<br />
alt="Number of Glycerolstocks" width="368"><p>Number of Glycerolstocks:<br /> 763 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/6/60/Freiburg10_Number_of_Team_members.png"<br />
alt="Number of Team members" width="368"><p>Number of Team members:<br /> 15 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/0/03/Freiburg10_Days_in_the_lab.png"<br />
alt="Days in the lab" width="368"><p>Days in the lab:<br /> 170 </p></div></li><br />
<li><div class="news"><img<br />
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_Longest_Labday.png"<br />
alt="Longest Labday" width="368"><p>Longest Labday:<br />18h </p></div></</li><br />
<li><div class="news"><img height="100px"<br />
src="https://static.igem.org/mediawiki/2010/b/b5/Freiburg10_Coffee_consumed_during_iGEM.png"<br />
alt="Coffee consumed during iGEM" width="184"><p>Coffee Consumed During iGEM:<br /> 2238 Cups</p></div></li><br />
<li><div class="news"><img heigth="100px"class="right" src="https://static.igem.org/mediawiki/2010/5/5f/Freiburg10_Virus_Logo_Small.png" id="ccc" /><p><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Statistics"><h5>!Freiburg Bioware Statistics!</h5></a></p></div></li><br />
</ul><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<div height="290px"><br />
<!---Box on the upper right: BioBricks---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><img class="right" src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg_10_BioBrick_icon_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><h2>BioBricks</h2></a><br />
<p class="standard">Modularization and modification of the viral capsids for retargeting approaches and directed gene delivery of suicide genes resulted in many different BioBricks.<br> Click here to explore them. <a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><span class="bold">[more]</a></span></p><br />
</div><br />
<br />
<!---Box on the lower left: Biosafety---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><img class="right" src="https://static.igem.org/mediawiki/2010/f/f9/Freiburg10_Danger_Virus_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><h2>Biosafety</h2></a><br />
<p class="standard">Aspects of biological safety were considered throughout and a risk assessment and security profile of the Adeno-associated Virus were created. Read more about Biosafety issues. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Safety"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<!---Box on the lower right: Notebook---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/95/Freiburg_10_Notebook_image_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
<h2>Notebook</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><span class="bold">From DNA to virus particle to cell culture? </span></a><br /><br />
Yes! Details on cloning, cell culture, flow cytometry analyses, quantifications by ELISA or Western Blot, cytotoxicity assays, time resolved microscopy ... <a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Methods"><span class="bold">[more]</span></a></p><br />
</div><br />
<br />
<br />
<!---Box on the lower right: Team---><br />
<div class="box_home_small"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_Virus_Logo_Small_small.png" id="ccc" /></a><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><br />
<h2>Team</h2></a><br />
<p><a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010"><span class="bold">Fun and Science at the same time?</span></a></a><br /><br />
Yes! The iGEM team Freiburg did not only share the lab bench, but as well some nice days <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Photo_Gallery"><span class="bold">canoeing</span></a> and initiated the photo contest Cuckoo Clock Competition. <a href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock"><span class="bold">[more]</span></a></p><br />
</div><br />
</div><br />
<div class="clearfix"></div><br />
<br /><br />
<h1>Sponsors</h1><br />
<br />
<div class="map" height="1169px"><br />
<map name="Sponsoren_iGEM" id="Sponsoren_iGEM"><br />
<area shape="circle" coords="705,369,65" href="http://www.uni-potsdam.de/" target="_blank" alt="Uni Potsdam" title="Uni Potsdam" /><br />
<area shape="circle" coords="156,138,121" href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank" alt="Uni Freiburg" title="Uni Freiburg" /><br />
<area shape="rect" coords="304,8,815,269" href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank" alt="Bioss" title="Bioss" /><br />
<area shape="rect" coords="501,311,611,434" href="http://www.molbiotech.uni-freiburg.de/" target="_blank" alt="kuk.lab" title="kuk.lab" /><br />
<area shape="rect" coords="274,329,480,412" href="http://www.daad.de/en/index.html" alt="daad" target="_blank" title="daad" /><br />
<area shape="rect" coords="67,324,238,419" href="http://www.frias.uni-freiburg.de/" alt="frias" target="_blank" title="frias" /><br />
<area shape="rect" coords="95,534,376,686" href="http://www.roche.com/index.htm" target="_blank" alt="Roche" title="Roche" /><br />
<area shape="rect" coords="510,458,677,606" href="http://www.qiagen.com/default_qs.aspx" target="_blank" alt="Qiagen" title="Qiagen" /><br />
<area shape="rect" coords="477,671,708,752" href="http://www.starlab.de/int/?l=2" target="_blank" alt="Starlab" title="Starlab" /><br />
<area shape="rect" coords="551,799,787,861" href="http://www.peqlab.com" target="_blank" alt="peqlab" title="peqlab" /><br />
<area shape="rect" coords="291,801,494,859" href="http://www.eppendorf.com/int/?l=1&amp;action=start" target="_blank" alt="Eppendorf" title="Eppendorf" /><br />
<area shape="rect" coords="43,801,226,859" href="http://www.gilson.com/en/" target="_blank" alt="Gilson" title="Gilson" /><br />
<area shape="rect" coords="44,872,218,936" href="http://www.fermentas.de/index.php?&amp;language=en" target="_blank" alt="Fermentas" title="Fermentas" /><br />
<area shape="rect" coords="42,942,259,1002" href="http://www.home.agilent.com" target="_blank" alt="aigilent" title="aigilent" /><br />
<area shape="rect" coords="289,958,388,1041" href="http://www.avidity.com/" target="_blank" alt="Avidity" title="Avidity" /><br />
<area shape="rect" coords="412,960,566,1048" href="http://www.hiss-dx.de/hiss/index.php?id=43&amp;L=1" target="_blank" alt="Hiss" title="Hiss" /><br />
<area shape="rect" coords="591,981,795,1043" href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank" alt="Biozym" title="Biozym" /><br />
<area shape="rect" coords="592,876,750,956" href="http://www.gatc-biotech.com" target="_blank" alt="GATC" title="GATC" /><br />
<area shape="rect" coords="296,886,540,948" href="http://www.geneart.com/" target="_blank" alt="Geneart" title="Geneart" /><br />
<area shape="rect" coords="495,1070,808,1151" href="http://www.atg-biosynthetics.com/" target="_blank" alt="ATG" title="ATG" /><br />
<area shape="rect" coords="242,1086,469,1154" href="http://www.purimex.com/" alt="purimex" target="_blank" title="purimex" /><br />
<area shape="rect" coords="45,1084,219,1154" href="http://www.dkfz.de/index.html" alt="dkfz" target="_blank" title="dkfz" /><br />
<area shape="rect" coords="46,1021,264,1075" href="http://www.mathworks.com" target="_blank" alt="Mathworks" title="Mathworks" /><br />
</map><br />
<img src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Sponsoren_Image_Map.gif" width="826" height="1169" border="0" alt="" title="" usemap="#Sponsoren_iGEM" /><br />
</div><br />
<br />
<br /><br />
<br /><br />
<center><a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" 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://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a></center><br />
<br /><br />
<center><small>primary team contact: Kristian M. Müller; <a href="mailto:Kristian@syntbio.net">e-mail: Kristian@syntbio.net</a></small></center><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Team/ContributionsTeam:Freiburg Bioware/Team/Contributions2010-10-27T19:16:41Z<p>Kristian: New page: Contributions</p>
<hr />
<div>Contributions</div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_PlasmidTeam:Freiburg Bioware/Project/Results/Modularization Vector Plasmid2010-10-25T16:39:53Z<p>Kristian: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoTocHeading">Contents</p><br />
<p class="MsoToc2"><span class="MsoHyperlink"><a<br />
href="#_Toc275794992">Modularization<br />
GOI/Vector Plasmid<span<br />
style="color: windowtext; display: none; text-decoration: none;">.<br />
</span><span<br />
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p><br />
<p class="MsoToc3"><span class="MsoHyperlink"><a<br />
href="#_Toc275794993">Introduction<br />
to Modularization<span<br />
style="color: windowtext; display: none; text-decoration: none;">.<br />
</span><span<br />
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p><br />
<p class="MsoToc3"><span class="MsoHyperlink"><a<br />
href="#_Toc275794994">Recombinant<br />
and Modular Vector Plasmid Carrying GOI<span<br />
style="color: windowtext; display: none; text-decoration: none;"><br />
</span><span<br />
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p><br />
<p class="MsoToc3"><span class="MsoHyperlink"><a<br />
href="#_Toc275794995">Cloning and<br />
Combination Strategies for the Vectorplasmid<span<br />
style="color: windowtext; display: none; text-decoration: none;">.<br />
</span><span<br />
style="color: windowtext; display: none; text-decoration: none;">2</span></a></span></p><br />
<p class="MsoToc3"><span class="MsoHyperlink"><a<br />
href="#_Toc275794996">Testing<br />
functionality of Assembled Vectorplasmid<span<br />
style="color: windowtext; display: none; text-decoration: none;">.<br />
</span><span<br />
style="color: windowtext; display: none; text-decoration: none;">3</span></a></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h2><a name="_Toc275794992">Modularization<br />
GOI/Vector Plasmid</a></h2><br />
<h3><a name="_Toc275794993">Introduction to<br />
Modularization</a></h3><br />
<p class="MsoNormal">Producing recombinant virus particles<br />
for therapeutical<br />
means is, besides specifically target cells, purification and<br />
quantification assays<br />
of AAV-2, one intention of the Virus Construction Kit provided by the<br />
iGEM team<br />
Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex<br />
components<br />
of AAV-2 were extracted and redesigned to match the iGEM standard.<br />
Functional<br />
activity was tested in cell culture.</p><br />
<p class="MsoNormal">Differing from the wildtype AAV-2<br />
genome, the Helper Free<br />
System provided by Stratagene comprises three plasmids and a<br />
specialized<br />
production cell line. AAV-293 cells derived from the HEK cell line<br />
express the<br />
stably integrated E1A and E1B helper proteins for efficient virus<br />
production.<br />
The plasmid containing the inverted terminal repeats (ITRs) is<br />
encapsidated<br />
into the preformed capsids after production of single-stranded DNA<br />
therefore<br />
also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i><br />
intron and the<br />
hGH terminator signal are flanked by the ITRs and serve in the host<br />
cell for<br />
regulation of transgene expression. In addition to that, the plasmid<br />
coding for<br />
the Rep and Cap proteins (pRC) can be provided <i>in trans</i><br />
leading to a layer<br />
of specificity due to the fact that the two genes are not packaged into<br />
the<br />
capsid since lacking of the ITRs impairs encapsidation. Another<br />
advantage of<br />
the Helper Free System can be attributed to cotransfection of another<br />
helper<br />
plasmid (pHelper), which provides the necessary proteins normally<br />
obtained by<br />
superinfection with helper viruses such as adenovirus or herpes simplex<br />
virus.<br />
These helper genes are required for full viral assembly by regulating<br />
gene<br />
expression of Rep and Cap proteins.</p><br />
<h3><a name="_Toc275794994">Recombinant and Modular<br />
Vector Plasmid Carrying GOI</a></h3><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010<br />
provides a modular Virus<br />
Construction Kit for therapeutical applications, quantification assays<br />
and<br />
purification approaches depending on capsid modifications and the gene<br />
of interest<br />
flanked by the inverted terminal repeats (ITRs. In order to produce<br />
BioBrick-compatible standardized biological parts, we reengineered the<br />
plasmids<br />
and added new components for gene therapy approaches and analysis of<br />
biological<br />
activity of assembled BioBrick parts. Each element required for intact<br />
and<br />
functional plasmids comprising the ITRs, a promoter, a putative<br />
enhancer<br />
element and the hGH terminator was PCR amplified and fused together <i>de<br />
novo</i>.<br />
As shown in Figure 1, the vectorplasmid was assembled with the produced<br />
BioBricks consisting of the left and right ITR (BBa_K404100 and<br />
BBa_K404101), a<br />
promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin<br />
intron<br />
(BBa_K404107), the gene of interests (fluorescent proteins mVenus:<br />
BBa_I757008 and<br />
mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39:<br />
BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</p><br />
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style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image001.gif"<br />
alt="Description: Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"<br />
height="83" width="102"></p><br />
</td><br />
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style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
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style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image002.gif"<br />
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height="69" width="86"></span></p><br />
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width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image003.gif"<br />
alt="Description: Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"<br />
height="78" width="116"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image004.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image004.gif"<br />
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width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"<br />
align="left"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image005.gif"<br />
alt="Description: Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"<br />
height="80" width="96"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image006.gif"<br />
alt="Description: Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"<br />
height="79" width="98"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image007.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image007.jpg"<br />
height="80" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Left ITR </span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(<span style="color: black;">BBa_K404100</span>)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image008.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image008.jpg"<br />
height="80" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">pCMV</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404102)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image009.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image009.jpg"<br />
height="79" width="78"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><i><span style="font-size: 9pt;"<br />
lang="DE">Beta-globin</span></i><span<br />
style="font-size: 9pt;" lang="DE"> intron </span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(<span style="color: black;">BBa_K404107</span>)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image010.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image010.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mVenus (BBa_I757008)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image011.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image011.jpg"<br />
height="80" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">hGH terminator</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404108)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image012.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image012.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Right ITR</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404101)</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image013.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image013.jpg"<br />
height="80" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">phTERT (BBa_K404106)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image014.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image014.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mCherry</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_J06504)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image015.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image015.jpg"<br />
height="81" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Cytosine deaminase (BBa_K404112)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image016.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image016.jpg"<br />
height="79" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mGMK_SR39</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404315)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image017.jpg"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image017.jpg"<br />
height="81" width="81"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mGMK_TK30</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404113)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="6"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image018.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image018.gif"<br />
height="116" width="555"></span></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="6"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275644946">Figure </a>1: <span<br />
style="font-weight: normal;">Overview of the theoretical<br />
sequence of each BioBrick provided within the Virus Construction Kit<br />
for an intact and fully functional rAAV genome. The plasmid in the<br />
lowest panel was used for tumor killing in combination with plasmids<br />
coding for modified capsid proteins. More detailed infomartion about<br />
these constructs can be found under ‘Arming: Killing the tumor’ and<br />
‘N-terminal fusion for Targeting’.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<h3><a name="_Toc275794995">Cloning and Combination<br />
Strategies for the<br />
Vectorplasmid</a> </h3><br />
<p class="MsoNormal">Organization of the recombinant viral<br />
DNA was modified ensuring<br />
several layers of specificity to our systems including a tumor-specific<br />
promoter and suicide genes encoding prodrug convertases. In order to<br />
modularize<br />
the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified<br />
and cloned<br />
into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team<br />
Freiburg_Bioware 2010 performed three site-directed mutagenesis in the<br />
gene of<br />
interest TK30 (BBa_K404109) and cytosine deaminase (<span<br />
style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)<br />
for deletion of PstI and NgoMIV<br />
iGEM site (for further information see the results page of ‘Arming –<br />
Killing<br />
the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich<br />
regions<br />
forming T-shaped hairpins during replication, PCR amplification was not<br />
possible. Hence a cloning strategy was developed by the iGEM team<br />
Freiburg in<br />
order to provide BioBrick-compatible ITRs (see ).</p><br />
<p class="MsoNormal">In Figure 2 the schematic overview of<br />
the modularization<br />
process can be seen which has been followed to conduct the assembly<br />
steps<br />
required for functional vectorplasmids.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 26.15pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"<br />
valign="top" width="454"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image019.gif"<br />
alt="Description: Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"<br />
height="272" width="439"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275783119">Figure </a>2: <span<br />
style="font-weight: normal;">Assembly procedure for fusion<br />
of BioBricks and composite parts to a fully assembled and functional<br />
plasmid coding for your gene of interest. This plasmid can be<br />
cotransfected with two helper plasmids providing protein for assembly<br />
and encapsidating of the rAAV genome (your gene of interest) into the<br />
capsids.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware<br />
provides two examples<br />
demonstrating the assembly procedure for constructing vectorplasmids.<br />
The first<br />
representative example is the fusion of the BioBrick part <i>beta-globin</i><br />
to<br />
the composite parts containing the 5´ elements of the plasmids, which<br />
are left<br />
ITR and CMV or phTERT promoter, respectively.</p><br />
<p class="MsoNormal" style="text-indent: 0cm;">As<br />
shown in Figure 3 the theoretical<br />
cloning performed for assembling the BioBricks <i>beta-globin </i>intron<br />
and<br />
leftITR_CMV together can be observed. </p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 23.95pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"<br />
valign="top" width="467"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image020.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image020.gif"<br />
height="530" width="452"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275783160">Figure </a>3: <span<br />
style="font-weight: normal;">Theoretical cloning of the<br />
composite part leftITR_CMV to the <i>beta-globin</i><br />
intron BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i><br />
intron.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<p class="MsoNormal">The plasmids were digested with both<br />
XbaI and PstI<br />
(beta-globin intron: <span<br />
style="font-size: 9pt; line-height: 200%; color: black;">BBa_K404107</span>)<br />
or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As<br />
demonstrated in<br />
the preparative gel in Figure 4, the expected bands could be detected<br />
under UV<br />
light and the extracted DNA could be successfully ligated. Each<br />
assembly step<br />
for producing BioBrick intermediates was conducted following the same<br />
strategy.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 129.95pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"<br />
valign="top" width="417"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="right"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image021.gif"<br />
alt="Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"<br />
height="194" width="397"></p><br />
<p class="MsoCaption"<br />
style="text-align: left; text-indent: 0cm;" align="left">Figure<br />
4: Assembly intermediate in fusion of the vectorplasmids containing<br />
different promoters. <span style="font-weight: normal;">Fusion<br />
of the BioBrick part <i>beta-globin</i> (</span><span<br />
style="color: black;">BBa_K404107</span><span<br />
style="font-weight: normal;">) intron to the composite parts<br />
leftITR_pCMV and leftITR_phTERT, respectively, was performed following<br />
the BioBrick assembly strategy by digesting the insert with PstI and<br />
XbaI and the vectors with SpeI and PstI. The left lane shows the<br />
expected fragment at around 560 bp which corresponds to the <i>beta-globin</i><br />
intron fragment, in contrast to the two lanes in the center and on the<br />
right which correspond to linearized plasmids after digesting with<br />
above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix;<br />
Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,<br />
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Separated fragments were extracted<br />
using the Gel Extraction<br />
Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase.<br />
After<br />
ligation has been carried out, <i>E. coli</i> XL-1B cells<br />
were transformed and incubated<br />
over night at 37°C. Picking clones from the transformation plate was<br />
performed<br />
the following day and DYT medium was inoculated incubating overnight.<br />
Plasmid<br />
DNA was isolated and test digestion revealed that cloning was<br />
successful<br />
obtaining the composite part leftITR_CMV_<i>beta-globin</i><br />
intron<br />
(BBa_K404117).</p><br />
<p class="MsoNormal">Plasmid production incorporating all<br />
required elements for<br />
transgene expression and genome encapsidation into empty viral capsids<br />
was<br />
performed by fusing the downstream elements consisting of the hGH<br />
terminator<br />
and right ITR to the intermediate part providing the gene of interest<br />
and the<br />
promoter fused to the left ITR. Figure 5 demonstrates the assembly<br />
performed<br />
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus<br />
and<br />
pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after<br />
digestion on the<br />
left lane fits to the hGH-terminator_rightITR length. The isolated<br />
fragments<br />
were ligated and successful assembly was confirmed by test digestion<br />
obtaining<br />
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i><br />
intron_mVenus_hGH_rightITR (<span style="color: black;">BBa_K404124</span>).<br />
</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 136.9pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"<br />
valign="top" width="411"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="right"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image022.gif"<br />
alt="Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"<br />
height="184" width="397"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784510">Figure </a>5<span<br />
style="font-weight: normal;">: </span>Modularization<br />
of the assembled vectorplasmid containing the phTERT promoter and<br />
mVenus as gene of interest.<span style="font-weight: normal;"><br />
Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus<br />
part&nbsp; to the composite parts pSB1C3_hGH_rightITR was performed<br />
following the BioBrick assembly strategy by digesting the insert with<br />
XbaI and PstI and the vector with SpeI and PstI. The left lane<br />
corresponds to linearized plasmid after digesting with above mentioned<br />
iGEM restriction sites whereas the right lane reveals an intensive band<br />
at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,<br />
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin<br />
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<p class="MsoNormal">Since cloning does not confirm<br />
biological activity, we<br />
analyzed the plasmids and their functional components, hGH terminator<br />
and <i>beta-globin</i><br />
intron, in cell culture. Assembled plasmids have been cotransfected,<br />
using<br />
AAV-293 cells, which provide the stable integrated E1A and E1B genes,<br />
with<br />
helper plasmids required for capsid assembly&nbsp; and genome<br />
encapsidation<br />
(pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus<br />
particles<br />
containing the single stranded DNA were harvested 72-hours post<br />
transfection<br />
and HT1080 cells transduced with constant volumes of viral vectors.<br />
48-hours<br />
post infection; transduced cells expressing the gene of interest were<br />
analyzed<br />
by flow cytometry. Facilitating and demonstrating the analysis of<br />
functionality<br />
of the assembled plasmid, mVenus was used in first place since<br />
fluorescent<br />
proteins enable facile visualization using fluorescent microscopy and<br />
flow<br />
cytometry analysis.</p><br />
<h3><a name="_Toc275794996">Testing functionality of<br />
Assembled Vectorplasmid</a></h3><br />
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span<br />
lang="DE">Fluorescence</span><br />
Microscopy of Target Cells Demonstrates GOI Expression</h4><br />
<p class="MsoNormal">Qualitative analysis of mVenus<br />
expression by fluorescence<br />
microscopy was conducted using Axio Observer Z1 showing that transduced<br />
HT1080<br />
cells and non-transduced cells could be easily distinguished. In Figure<br />
6 cells<br />
were excited with 505nm and fluorescence emission at 536nm was<br />
detected.<br />
Therefore, successful infection of tumor cells by recombinant viral<br />
particles<br />
carrying the assembled vectorplasmid coding for mVenus could be<br />
demonstrated. </p><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"<br />
valign="top" width="334"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">A</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image023.jpg"<br />
alt="Description: Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"<br />
height="218" width="264"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"<br />
valign="top" width="307"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">B</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image024.jpg"<br />
alt="Description: Description: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"<br />
height="220" width="242"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"<br />
valign="top" width="334"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">C</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image025.jpg"<br />
alt="Description: Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"<br />
height="195" width="258"></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"<br />
valign="top" width="307"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">D</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image026.jpg"<br />
alt="Description: Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"<br />
height="194" width="257"></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="2"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784524">Figure </a>6: <span<br />
style="font-weight: normal;">Fluorescence microscopy<br />
(Exciatation: 505nm, Emission: 536nm) was performed for detection of<br />
transduced cell expression mVenus. A:Cells detected in bright field<br />
picture B: Detection of mVenus expression can be observed.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span<br />
lang="DE">Analysis </span>of<br />
Target Cells by Flow Cytometry demonstrates GOI Expression</h4><br />
<p class="MsoNormal">Characterizing the function of the<br />
hGH terminator, the <i>beta-globin</i><br />
intron and the complete plasmid, several approaches were conducted<br />
followed by analysis<br />
via flow cytometry. </p><br />
<h5>Influence of hGH terminator BioBrick on GOI Expression</h5><br />
<p class="MsoNormal">The iGEM team Freiburg provides the<br />
hGH plolyadenylation<br />
sequence within the ‘Virus Construction Kit’ due to the fact that<br />
almost every<br />
eukaryotic mRNA is processed at their 3´ and 5´end except for histone<br />
mRNAs<br />
(Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved<br />
sequences.<br />
First, the polyadenylation signal “AATAAA” which is recognized by the<br />
multiprotein complex and second the GT-rich region (downstream sequence<br />
element, DSE) which is located 30 nucleotides downstream of the<br />
cleavage site.<br />
The assembled 3´end-processing machinery cleaves the mRNA transcript<br />
immediately after a CA-nucleotide therefore defining the cleavage site<br />
(Danckwardt et al. 2008)<span<br />
style="font-size: 12pt; line-height: 200%;">. </span>Recombinant<br />
vectorplasmids were engineered containing the inverted terminal repeats<br />
(ITRs),<br />
a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as<br />
gene of<br />
interest with and without the hGH terminator signal. Transduction of<br />
HT1080<br />
cells with constant volume of viral particles containing the<br />
vectorplasmids and<br />
measuring mVenus expression 24-hours post infection by flow cytometry<br />
demonstrated that transgene expression of the constructs lacking the<br />
hGH<br />
termination signal is significantly reduced as shown in Figure 7 and<br />
Figure 8<br />
confirming the expected results that hGH is essential for mRNA<br />
processing. The<br />
iGEM team Freiburg_Bioware 2010 therefore suggests using the provided<br />
hGH<br />
termination signal within the Virus Construction Kit for optimal gene<br />
expression.</p><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
lacking hGH termination signal</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image027.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image027.gif"<br />
height="408" width="629"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
containing hGH terminator signal</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image028.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image028.gif"<br />
height="410" width="636"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a<br />
name="_Ref275784539"><b>Figure </b></a><b>7:</b><br />
<b>Flow cytometry analysis of vectorplasmids with and<br />
without hGH terminator.</b> </p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>A</b>:<br />
Gating non transduced cells (control); subcellular debris and clumps<br />
can be distinguished from single cells by size, estimated forward<br />
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) <b>B</b>:<br />
Non transduced cells applied against mVenus (Analytical gate was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region (R5). <b>C</b>: Gating transduced cells<br />
(R2 <span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)<br />
(used plasmids for transfection: GOI: <b>pSB1C3_lITR_CMV_beta-globin<br />
intron_mVenus_rITR (BBa_K404127)</b>, pHelper, pRC). <b>D</b>:<br />
Transduced cells plotted against mVenus, R10 comprises transduced cells<br />
by detecting mVenus expression. <b>E</b>: Overlay of<br />
non-transduced (red) and transduced (green) cells applied against<br />
mVenus.<b>F</b>: Gating non-transduced cells (control) <b>G</b>:<br />
Non-transduced cells applied against mVenus. <b>H</b>:<br />
Gating transduced cells (R2 <span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)<br />
(used plasmids for transfection: GOI: reassembled <b>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(BBa_K404119)</b>, pHelper, pRC). <b>I</b>:<br />
Transduced cells applied against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression. <b>J</b>: Overlay<br />
of non-transduced (red) and transduced (green) cells applied against<br />
mVenus.</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<img src="Freiburg10_Modularization_GOI_02_files/image029.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image029.gif"<br />
height="355" width="473"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784545">Figure </a>8: Flow cytometry<br />
analysis of vectorplasmids with and without hGH terminator.<span<br />
style="font-weight: normal;"> YFP expression of viral<br />
genomes was determined by flow cytomery after 24-hour post infection.<br />
Results demonstrate that mVenus expression of vectorplasmids lacking<br />
the hGH terminator is reduced significantly proving that the<br />
polyadenylation signal is essential for viral gene expression using<br />
recombinant viral vectors engineered by using components of the Virus<br />
Construction Kit.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoCaption">&nbsp;</p><br />
<h5>Influence of <i>Beta-globin</i> intron Biobrick<br />
on GOI Expression</h5><br />
<p class="MsoNormal">Providing an element assumed to be an<br />
enhancer of transgene<br />
expression (Nott et al. 2003), the iGEM team Freiburg tested a<br />
beta-globin<br />
intron derived from the human <i>beta globin</i> gene<br />
which can be fused<br />
upstream of the desired gene of interest. The beta-globin intron<br />
BioBrick<br />
consists of a partial chimeric CMV promoter followed by the intron II<br />
of the <i>beta-globin</i><br />
gene. The 3´end of the intron is fused to the first 25 bases of human <i>beta<br />
globin</i> gene exon 3. The <i>beta globin</i><br />
intron BioBrick is assumed to<br />
enhance eukaryotic gene expression (Nott et al. 2003). Analysis was<br />
conducted<br />
as described for the hGH terminator experiment (see above). As shown in<br />
Figure<br />
9 and Figure 10 the vectorplasmid missing the <i>beta-globin</i><br />
intron showed<br />
a negligible difference in mVenus expression compared to viral genomes<br />
containing the <i>beta-globin</i> intron. Considering<br />
these results and taking<br />
into account that a constant volume of viral particles has been used<br />
for<br />
transduction, the difference between the construct containing and<br />
lacking the<br />
beta-globin intron is minimal. Since packaging efficiency of the AAV-2<br />
decreases with increasing sizes of the insert (Dong et al. 1996), the<br />
iGEM team<br />
Freiburg_Bioware suggests using the <i>beta-globin </i>intron<br />
in dependence on<br />
the size of your transgene.</p><br />
<table class="MsoNormalTable"<br />
style="width: 490.75pt; border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0" width="654"><br />
<tbody><br />
<tr style="height: 2.5pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
lacking <i>beta-globin</i> intron</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image030.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image030.gif"<br />
height="412" width="640"></p><br />
</td><br />
</tr><br />
<tr style="height: 106.5pt;"><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
containing <i>beta-globin</i> intron</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image031.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image031.gif"<br />
height="410" width="636"></p><br />
</td><br />
</tr><br />
<tr style="height: 106.5pt;"><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784803">Figure </a>9: Flow cytometry<br />
analysis of vectorplasmids with and without <i>beta-globin</i><br />
intron.&nbsp; A<span style="font-weight: normal;">:<br />
Gating non transduced cells (control); subcellular debris and clumps<br />
can be distinguished from single cells by size, estimated forward<br />
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) </span>B<span<br />
style="font-weight: normal;">: Non transduced cells applied<br />
against mVenus (Analytical gate was set such that 1% or fewer of<br />
negative control cells fell within the positive region (R5). </span>C<span<br />
style="font-weight: normal;">: Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: GOI: </span>pSB1C3_lITR_CMV_mVenus_hGH_rITR<br />
(BBa_K404128)<span style="font-weight: normal;">, pHelper,<br />
pRC). </span>D<span style="font-weight: normal;">:<br />
Transduced cells plotted against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression </span>E<span<br />
style="font-weight: normal;">: Overlay of non-transduced<br />
(red) and transduced (green) cells applied against mVenus </span>F<span<br />
style="font-weight: normal;">: Gating non-transduced cells<br />
(control). </span>G<span style="font-weight: normal;">:<br />
Non-transduced cells applied against mVenus (R5).</span>H<span<br />
style="font-weight: normal;">: Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: GOI: reassembled </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(BBa_K404119)<span style="font-weight: normal;">, pHelper,<br />
pRC). </span>I<span style="font-weight: normal;">:<br />
Transduced cells applied against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression. </span>J<span<br />
style="font-weight: normal;">: Overlay of non-transduced<br />
(red) and transduced (green) cells applied against mVenus.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoNormalTable"<br />
style="width: 490.75pt; border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0" width="654"><br />
<tbody><br />
<tr style="height: 90.2pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"<br />
align="left">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image032.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image032.gif"<br />
height="332" width="450"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784805">Figure </a>10: Flow cytometry<br />
analysis of vectorplasmids with and without <i>beta-globin</i><br />
intron.<span style="font-weight: normal;"> 48-hours post<br />
transfection, viral particles were harvested by freeze-thaw lysis and<br />
centrifugation followed by HT1080 transduction. YFP expression of<br />
vectorplasmids was determined by flow cytometry 24-hours post<br />
infection. The vectorplasmid missing the beta-globin intron showed a<br />
negligible difference in mVenus expression compared to viral plasmid<br />
containing the beta-globin intron.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<h5>Functionality of the Full Assembled Vectorplasmid<br />
Demonstrated by GOI<br />
Expression </h5><br />
<p class="MsoNormal">After assembly of plasmids containing<br />
all required elements<br />
(see Figure 1), functionality was tested in cell culture. AAV-293 cells<br />
stably<br />
expressing E1A and E1B proteins were transfected with three plasmids<br />
&nbsp;(pHelper, pRC, pGOI). Virus particles were harvested 72-hours<br />
post-transfection and the tumor cell line HT1080 was transduced with<br />
the<br />
recombinant viral vectors encapsidating the gene of interest mVenus<br />
(BBa_I757008).</p><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010<br />
compared the<br />
standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived<br />
from the<br />
Stratagene system) with the assembled plasmid<br />
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus:<br />
BBa_K404119).<br />
Fluorescence expression data obtained by flow cytometry analysis are<br />
shown in<br />
Figure 11 and Figure 12. Comparing mVenus expression of the standard<br />
plasmid<br />
and the modified, assembled plasmid reveals that biological<br />
functionality of<br />
the reassembled plasmid was confirmed. </p><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span<br />
lang="DE">pSB1C3_mVenus (BBa_K404119)</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image033.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image033.gif"<br />
height="409" width="634"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pAAV_mVenus<br />
(Stratagene)</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image034.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image034.gif"<br />
height="406" width="630"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784576">Figure </a>11: Flow cytometry<br />
analysis of reassembled vectorplasmid (BBa_K404119) compared to<br />
standard plasmid provided by Stratagene. A<span<br />
style="font-weight: normal;">: Gating non transduced cells<br />
(control); subcellular debris and clumps can be distinguished from<br />
single cells by size, estimated forward scatter (FS Lin) and<br />
granularity, estimated side scatter (SS Lin) B: Non transduced cells<br />
plotted against mVenus (Analytical gate was set such that 1% or fewer<br />
of negative control cells fell within the positive region (R5).C:<br />
Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: pGOI: </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(pSB1C3_mVenus: BBa_K404119<span<br />
style="color: rgb(0, 176, 80); font-weight: normal;">)</span><span<br />
style="font-weight: normal;">, pHelper, pRC. </span>D<span<br />
style="font-weight: normal;">: Transduced cells plotted<br />
against mVenus, R10 comprised transduced cells, by detecting mVenus<br />
expression. </span>E<span style="font-weight: normal;">:<br />
Overlay of non-transduced (red) and transduced (green). </span>F<span<br />
style="font-weight: normal;">: Gating non transduced cells<br />
(control). </span>G<span style="font-weight: normal;">:<br />
Non-transduced cells plotted against mVenus (R5). </span>H<span<br />
style="font-weight: normal;">: Gating transduced cells (R14 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R2) (used plasmids for<br />
transfection: pGOI: pAAV_mVenus, pHelper). </span>I<span<br />
style="font-weight: normal;">: Transduced cells plotted<br />
against mVenus, R10 comprised transduced cells, by detecting mVenus<br />
expression.</span> J<span style="font-weight: normal;">:<br />
Overlay of non-transduced (red) and transduced (green) cells plotted<br />
against mVenus expression. </span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoNormalTable"<br />
style="border-collapse: collapse;" border="0"<br />
cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><img<br />
src="Freiburg10_Modularization_GOI_02_files/image035.gif"<br />
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image035.gif"<br />
height="396" width="541"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784852">Figure </a>12: Flow cytometry<br />
analysis of reassembled vectorplasmid (BBa_K404119) compared to<br />
standard plasmid provided by Stratagene. <span<br />
style="font-weight: normal;">Fluorescence of the standard<br />
plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus<br />
(BBa_K404119) construct was measured. As demonstrated mVenus expression<br />
is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the<br />
standard pAAV_mVenus construct.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal">&nbsp;</p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Danckwardt, S.,<br />
Hentze, M.W. &amp; Kulozik, A.E., 2008. 3’ end mRNA processing:<br />
molecular<br />
mechanisms and implications for health and disease. <i>The EMBO<br />
journal</i>,<br />
27(3), pp.482-98. Available at:<br />
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Dong, J.Y., Fan,<br />
P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the<br />
packaging capacity<br />
of recombinant adeno-associated virus. <i>Human gene therapy</i>,<br />
7(17),<br />
pp.2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Millevoi, S. et<br />
al., 2006. An interaction between U2AF 65 and CF I(m) links the<br />
splicing and 3’<br />
end processing machineries. <i>The EMBO journal</i>,<br />
25(20), pp.4854-64.<br />
Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Nott, A., Meislin,<br />
S.H. &amp; Moore, M.J., 2003. A quantitative analysis of intron<br />
effects on<br />
mammalian gene expression. <i>RNA (New York, N.Y.)</i>,<br />
9(5), pp.607-17.<br />
Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p><br />
<br />
</html></div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_PlasmidTeam:Freiburg Bioware/Project/Results/Modularization Vector Plasmid2010-10-25T16:12:48Z<p>Kristian: New page: {{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}} <html> <h2>Modularization GOI/Vector Plasmid</h2> <h3>Introduction to Modularization</h...</p>
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<html><br />
<h2>Modularization GOI/Vector Plasmid</h2><br />
<h3>Introduction to Modularization</h3><br />
<p class="MsoNormal">Producing recombinant virus particles<br />
for therapeutical<br />
means is, besides specifically target cells, purification and<br />
quantification<br />
assays of AAV-2, one intention of the Virus Construction Kit provided<br />
by the<br />
iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the<br />
complex<br />
components of AAV-2 were extracted and redesigned to match the iGEM<br />
standard.<br />
Functional activity was tested in cell culture.</p><br />
<p class="MsoNormal">Differing from the wildtype AAV-2<br />
genome, the Helper Free<br />
System provided by Stratagene comprises three plasmids and a<br />
specialized<br />
production cell line. AAV-293 cells derived from the HEK cell line<br />
express the<br />
stably integrated E1A and E1B helper proteins for efficient virus<br />
production. The<br />
plasmid containing the inverted terminal repeats (ITRs) is encapsidated<br />
into<br />
the preformed capsids after production of single-stranded DNA therefore<br />
also<br />
known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i><br />
intron and the hGH<br />
terminator signal are flanked by the ITRs and serve in the host cell<br />
for<br />
regulation of transgene expression. In addition to that, the plasmid<br />
coding for<br />
the Rep and Cap proteins (pRC) can be provided <i>in trans</i><br />
leading to a<br />
layer of specificity due to the fact that the two genes are not<br />
packaged into<br />
the capsid since lacking of the ITRs impairs encapsidation. Another<br />
advantage<br />
of the Helper Free System can be attributed to cotransfection of<br />
another helper<br />
plasmid (pHelper), which provides the necessary proteins normally<br />
obtained by<br />
superinfection with helper viruses such as adenovirus or herpes simplex<br />
virus.<br />
These helper genes are required for full viral assembly by regulating<br />
gene<br />
expression of Rep and Cap proteins.</p><br />
<h3>Recombinant and Modular Vector Plasmid Carrying GOI</h3><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010<br />
provides a modular Virus<br />
Construction Kit for therapeutical applications, quantification assays<br />
and<br />
purification approaches depending on capsid modifications and the gene<br />
of<br />
interest flanked by the inverted terminal repeats (ITRs. In order to<br />
produce<br />
BioBrick-compatible standardized biological parts, we reengineered the<br />
plasmids<br />
and added new components for gene therapy approaches and analysis of<br />
biological<br />
activity of assembled BioBrick parts. Each element required for intact<br />
and<br />
functional plasmids comprising the ITRs, a promoter, a putative<br />
enhancer<br />
element and the hGH terminator was PCR amplified and fused together <i>de<br />
novo</i>.<br />
As shown in Figure 1, the vectorplasmid was assembled with the produced<br />
BioBricks consisting of the left and right ITR (BBa_K404100 and<br />
BBa_K404101), a<br />
promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin<br />
intron (BBa_K404107),<br />
the gene of interests (fluorescent proteins mVenus: BBa_I757008 and<br />
mCherry: BBa_J06504,<br />
suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD:<br />
BBa_K404112)<br />
and the hGH terminator (BBa_K404108).</p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 1"<br />
src="Freiburg10_Modularization_GOI_02_files/image001.gif"<br />
alt="Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"<br />
height="83" width="102"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Grafik 51"<br />
src="Freiburg10_Modularization_GOI_02_files/image002.gif"<br />
height="69" width="86"></span></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 6"<br />
src="Freiburg10_Modularization_GOI_02_files/image003.gif"<br />
alt="Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"<br />
height="78" width="116"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 26"<br />
src="Freiburg10_Modularization_GOI_02_files/image004.gif"<br />
height="66" width="90"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"<br />
align="left"><img id="Grafik 8"<br />
src="Freiburg10_Modularization_GOI_02_files/image005.gif"<br />
alt="Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"<br />
height="80" width="96"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 4"<br />
src="Freiburg10_Modularization_GOI_02_files/image006.gif"<br />
alt="Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"<br />
height="79" width="98"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 43"<br />
src="Freiburg10_Modularization_GOI_02_files/image007.jpg"<br />
height="80" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Left ITR </span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(</span><span<br />
style="font-size: 9pt; color: black;" lang="DE">BBa_K404100</span><span<br />
style="font-size: 9pt;" lang="DE">)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 70"<br />
src="Freiburg10_Modularization_GOI_02_files/image008.jpg"<br />
height="80" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">pCMV</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404102)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 61"<br />
src="Freiburg10_Modularization_GOI_02_files/image009.jpg"<br />
height="79" width="78"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><i><span style="font-size: 9pt;"<br />
lang="DE">Beta-globin</span></i><span<br />
style="font-size: 9pt;" lang="DE"> intron </span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(</span><span<br />
style="font-size: 9pt; color: black;" lang="DE">BBa_K404107</span><span<br />
style="font-size: 9pt;" lang="DE">)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 58"<br />
src="Freiburg10_Modularization_GOI_02_files/image010.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mVenus (BBa_I757008)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 64"<br />
src="Freiburg10_Modularization_GOI_02_files/image011.jpg"<br />
height="80" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">hGH terminator</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404108)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 46"<br />
src="Freiburg10_Modularization_GOI_02_files/image012.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Right ITR</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404101)</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 67"<br />
src="Freiburg10_Modularization_GOI_02_files/image013.jpg"<br />
height="80" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">phTERT (BBa_K404106)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 109"<br />
src="Freiburg10_Modularization_GOI_02_files/image014.jpg"<br />
height="79" width="79"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mCherry</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_J06504)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 10"<br />
src="Freiburg10_Modularization_GOI_02_files/image015.jpg"<br />
height="81" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">Cytosine deaminase (BBa_K404112)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 13"<br />
src="Freiburg10_Modularization_GOI_02_files/image016.jpg"<br />
height="79" width="80"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mGMK_SR39</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404315)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"<br />
width="109"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 76.9pt;"<br />
width="103"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 91.7pt;"<br />
width="122"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 73.75pt;"<br />
width="98"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Picture 7"<br />
src="Freiburg10_Modularization_GOI_02_files/image017.jpg"<br />
height="81" width="81"></span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">mGMK_TK30</span></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">(BBa_K404113)</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 77.75pt;"<br />
width="104"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 79.05pt;"<br />
width="105"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><span style="font-size: 9pt;"<br />
lang="DE">&nbsp;</span></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="6"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><span style="font-size: 9pt;"><img<br />
id="Grafik 2057"<br />
src="Freiburg10_Modularization_GOI_02_files/image018.gif"<br />
height="116" width="555"></span></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="6"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275644946">Figure </a>1: <span<br />
style="font-weight: normal;">Overview of the theoretical<br />
sequence of each BioBrick provided within the Virus Construction Kit<br />
for an intact and fully functional rAAV genome. The plasmid in the<br />
lowest panel was used for tumor killing in combination with plasmids<br />
coding for modified capsid proteins. More detailed infomartion about<br />
these constructs can be found under ‘Arming: Killing the tumor’ and<br />
‘N-terminal fusion for Targeting’.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<h3>Cloning and Combination Strategies for the Vectorplasmid </h3><br />
<p class="MsoNormal">Organization of the recombinant viral<br />
DNA was modified<br />
ensuring several layers of specificity to our systems including a<br />
tumor-specific promoter and suicide genes encoding prodrug convertases.<br />
In<br />
order to modularize the rAAV sequence, each plasmid element (Figure 1)<br />
was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3.<br />
Furthermore, the iGEM<br />
team Freiburg_Bioware 2010 performed three site-directed mutagenesis in<br />
the<br />
gene of interest TK30 (BBa_K404109) and cytosine deaminase (<span<br />
style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)<br />
for deletion of<br />
PstI and NgoMIV iGEM site (for further information see the results page<br />
of<br />
‘Arming – Killing the tumor’). Since the inverted terminal repeats<br />
(ITRs) are<br />
GC-rich regions forming T-shaped hairpins during replication, PCR<br />
amplification<br />
was not possible. Hence a cloning strategy was developed by the iGEM<br />
team<br />
Freiburg in order to provide BioBrick-compatible ITRs (see ).</p><br />
<p class="MsoNormal">In Figure 2 the schematic overview of<br />
the modularization<br />
process can be seen which has been followed to conduct the assembly<br />
steps<br />
required for functional vectorplasmids.</p><br />
<div align="center"><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 26.15pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"<br />
valign="top" width="454"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
id="Grafik 2"<br />
src="Freiburg10_Modularization_GOI_02_files/image019.gif"<br />
alt="Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"<br />
height="272" width="439"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275783119">Figure </a>2: <span<br />
style="font-weight: normal;">Assembly procedure for fusion<br />
of BioBricks and composite parts to a fully assembled and functional<br />
plasmid coding for your gene of interest. This plasmid can be<br />
cotransfected with two helper plasmids providing protein for assembly<br />
and encapsidating of the rAAV genome (your gene of interest) into the<br />
capsids.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware<br />
provides two examples<br />
demonstrating the assembly procedure for constructing vectorplasmids.<br />
The first<br />
representative example is the fusion of the BioBrick part <i>beta-globin</i><br />
to<br />
the composite parts containing the 5´ elements of the plasmids, which<br />
are left<br />
ITR and CMV or phTERT promoter, respectively.</p><br />
<p class="MsoNormal" style="text-indent: 0cm;">As<br />
shown in Figure 3 the theoretical<br />
cloning performed for assembling the BioBricks <i>beta-globin </i>intron<br />
and<br />
leftITR_CMV together can be observed. </p><br />
<div align="center"><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 23.95pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"<br />
valign="top" width="467"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
id="Grafik 74"<br />
src="Freiburg10_Modularization_GOI_02_files/image020.gif"<br />
height="530" width="452"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275783160">Figure </a>3: <span<br />
style="font-weight: normal;">Theoretical cloning of the<br />
composite part leftITR_CMV to the <i>beta-globin</i><br />
intron BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i><br />
intron.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<p class="MsoNormal">The plasmids were digested with both<br />
XbaI and PstI<br />
(beta-globin intron: <span<br />
style="font-size: 9pt; line-height: 200%; color: black;">BBa_K404107</span>)<br />
or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As<br />
demonstrated in<br />
the preparative gel in Figure 4, the expected bands could be detected<br />
under UV<br />
light and the extracted DNA could be successfully ligated. Each<br />
assembly step<br />
for producing BioBrick intermediates was conducted following the same<br />
strategy.</p><br />
<div align="center"><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 129.95pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"<br />
valign="top" width="417"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="right"><img id="Grafik 77"<br />
src="Freiburg10_Modularization_GOI_02_files/image021.gif"<br />
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"<br />
height="194" width="397"></p><br />
<p class="MsoCaption"<br />
style="text-align: left; text-indent: 0cm;" align="left">Figure<br />
4: Assembly intermediate in fusion of the vectorplasmids containing<br />
different promoters. <span style="font-weight: normal;">Fusion<br />
of the BioBrick part <i>beta-globin</i> (</span><span<br />
style="color: black;">BBa_K404107</span><span<br />
style="font-weight: normal;">) intron to the composite parts<br />
leftITR_pCMV and leftITR_phTERT, respectively, was performed following<br />
the BioBrick assembly strategy by digesting the insert with PstI and<br />
XbaI and the vectors with SpeI and PstI. The left lane shows the<br />
expected fragment at around 560 bp which corresponds to the <i>beta-globin</i><br />
intron fragment, in contrast to the two lanes in the center and on the<br />
right which correspond to linearized plasmids after digesting with<br />
above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix;<br />
Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,<br />
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Separated fragments were extracted<br />
using the Gel Extraction<br />
Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase.<br />
After<br />
ligation has been carried out, <i>E. coli</i> XL-1B cells<br />
were transformed and<br />
incubated over night at 37°C. Picking clones from the transformation<br />
plate was<br />
performed the following day and DYT medium was inoculated incubating<br />
overnight.<br />
Plasmid DNA was isolated and test digestion revealed that cloning was<br />
successful obtaining the composite part leftITR_CMV_<i>beta-globin</i><br />
intron (BBa_K404117).</p><br />
<p class="MsoNormal">Plasmid production incorporating all<br />
required elements for<br />
transgene expression and genome encapsidation into empty viral capsids<br />
was<br />
performed by fusing the downstream elements consisting of the hGH<br />
terminator and<br />
right ITR to the intermediate part providing the gene of interest and<br />
the<br />
promoter fused to the left ITR. Figure 5 demonstrates the assembly<br />
performed<br />
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus<br />
and<br />
pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after<br />
digestion on the<br />
left lane fits to the hGH-terminator_rightITR length. The isolated<br />
fragments<br />
were ligated and successful assembly was confirmed by test digestion<br />
obtaining<br />
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i><br />
intron_mVenus_hGH_rightITR<br />
(<span style="line-height: 200%; color: black;">BBa_K404124</span>).<br />
</p><br />
<div align="center"><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr style="height: 136.9pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"<br />
valign="top" width="411"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="right"><img id="Grafik 80"<br />
src="Freiburg10_Modularization_GOI_02_files/image022.gif"<br />
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"<br />
height="184" width="397"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784510">Figure </a>5<span<br />
style="font-weight: normal;">: </span>Modularization<br />
of the assembled vectorplasmid containing the phTERT promoter and<br />
mVenus as gene of interest.<span style="font-weight: normal;"><br />
Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus<br />
part&nbsp; to the composite parts pSB1C3_hGH_rightITR was performed<br />
following the BioBrick assembly strategy by digesting the insert with<br />
XbaI and PstI and the vector with SpeI and PstI. The left lane<br />
corresponds to linearized plasmid after digesting with above mentioned<br />
iGEM restriction sites whereas the right lane reveals an intensive band<br />
at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,<br />
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin<br />
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<p class="MsoNormal">Since cloning does not confirm<br />
biological activity, we<br />
analyzed the plasmids and their functional components, hGH terminator<br />
and <i>beta-globin</i><br />
intron, in cell culture. Assembled plasmids have been cotransfected,<br />
using<br />
AAV-293 cells, which provide the stable integrated E1A and E1B genes,<br />
with<br />
helper plasmids required for capsid assembly&nbsp; and genome<br />
encapsidation (pRC and<br />
pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles<br />
containing the single stranded DNA were harvested 72-hours post<br />
transfection<br />
and HT1080 cells transduced with constant volumes of viral vectors.<br />
48-hours<br />
post infection; transduced cells expressing the gene of interest were<br />
analyzed<br />
by flow cytometry. Facilitating and demonstrating the analysis of<br />
functionality<br />
of the assembled plasmid, mVenus was used in first place since<br />
fluorescent<br />
proteins enable facile visualization using fluorescent microscopy and<br />
flow<br />
cytometry analysis.</p><br />
<h3>Testing functionality of Assembled Vectorplasmid</h3><br />
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span<br />
lang="DE">Fluorescence</span><br />
Microscopy of Target Cells Demonstrates GOI Expression</h4><br />
<p class="MsoNormal">Qualitative analysis of mVenus<br />
expression by fluorescence<br />
microscopy was conducted using Axio Observer Z1 showing that transduced<br />
HT1080<br />
cells and non-transduced cells could be easily distinguished. In Figure<br />
6 cells were excited with 505nm and fluorescence emission at 536nm was<br />
detected. Therefore, successful infection of tumor cells by recombinant<br />
viral<br />
particles carrying the assembled vectorplasmid coding for mVenus could<br />
be<br />
demonstrated. </p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"<br />
valign="top" width="334"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">A</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 18"<br />
src="Freiburg10_Modularization_GOI_02_files/image023.jpg"<br />
alt="Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"<br />
height="218" width="264"></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"<br />
valign="top" width="307"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">B</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 19"<br />
src="Freiburg10_Modularization_GOI_02_files/image024.jpg"<br />
alt="Description: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"<br />
height="220" width="242"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"<br />
valign="top" width="334"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">C</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 16"<br />
src="Freiburg10_Modularization_GOI_02_files/image025.jpg"<br />
alt="Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"<br />
height="195" width="258"></p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"<br />
valign="top" width="307"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">D</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"<br />
align="center"><img id="Grafik 17"<br />
src="Freiburg10_Modularization_GOI_02_files/image026.jpg"<br />
alt="Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"<br />
height="194" width="257"></p><br />
</td><br />
</tr><br />
<tr><br />
<td colspan="2"<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784524">Figure </a>6: <span<br />
style="font-weight: normal;">Fluorescence microscopy<br />
(Exciatation: 505nm, Emission: 536nm) was performed for detection of<br />
transduced cell expression mVenus. A:Cells detected in bright field<br />
picture B: Detection of mVenus expression can be observed.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span<br />
lang="DE">Analysis</span><span lang="DE"><br />
</span>of Target Cells by Flow Cytometry demonstrates GOI<br />
Expression</h4><br />
<p class="MsoNormal">Characterizing the function of the<br />
hGH terminator, the <i>beta-globin</i><br />
intron and the complete plasmid, several approaches were conducted<br />
followed by<br />
analysis via flow cytometry. </p><br />
<h5>Influence of hGH terminator BioBrick on GOI Expression</h5><br />
<p class="MsoNormal">The iGEM team Freiburg provides the<br />
hGH plolyadenylation<br />
sequence within the ‘Virus Construction Kit’ due to the fact that<br />
almost every<br />
eukaryotic mRNA is processed at their 3´ and 5´end except for histone<br />
mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved<br />
sequences. First, the<br />
polyadenylation signal “AATAAA” which is recognized by the multiprotein<br />
complex<br />
and second the GT-rich region (downstream sequence element, DSE) which<br />
is<br />
located 30 nucleotides downstream of the cleavage site. The assembled<br />
3´end-processing machinery cleaves the mRNA transcript immediately<br />
after a<br />
CA-nucleotide therefore defining the cleavage site (Danckwardt et al.<br />
2008)<span style="font-size: 12pt; line-height: 200%;">. </span>Recombinant<br />
vectorplasmids were engineered containing the inverted terminal repeats<br />
(ITRs),<br />
a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as<br />
gene of<br />
interest with and without the hGH terminator signal. Transduction of<br />
HT1080<br />
cells with constant volume of viral particles containing the<br />
vectorplasmids and<br />
measuring mVenus expression 24-hours post infection by flow cytometry<br />
demonstrated that transgene expression of the constructs lacking the<br />
hGH<br />
termination signal is significantly reduced as shown in Figure 7 and<br />
Figure 8 confirming the expected results that hGH is essential for mRNA<br />
processing.<br />
The iGEM team Freiburg_Bioware 2010 therefore suggests using the<br />
provided hGH<br />
termination signal within the Virus Construction Kit for optimal gene<br />
expression.</p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
lacking hGH termination signal</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
id="Grafik 30"<br />
src="Freiburg10_Modularization_GOI_02_files/image027.gif"<br />
height="408" width="629"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
containing hGH terminator signal</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
id="Grafik 2049"<br />
src="Freiburg10_Modularization_GOI_02_files/image028.gif"<br />
height="410" width="636"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a<br />
name="_Ref275784539"><b>Figure </b></a><b>7</b><b>:</b><br />
<b>Flow cytometry analysis of vectorplasmids with and<br />
without hGH terminator.</b> </p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>A</b>:<br />
Gating non transduced cells (control); subcellular debris and clumps<br />
can be distinguished from single cells by size, estimated forward<br />
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) <b>B</b>:<br />
Non transduced cells applied against mVenus (Analytical gate was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region (R5). <b>C</b>: Gating transduced cells<br />
(R2 <span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)<br />
(used plasmids for transfection: GOI: <b>pSB1C3_lITR_CMV_beta-globin<br />
intron_mVenus_rITR (BBa_K404127)</b>, pHelper, pRC). <b>D</b>:<br />
Transduced cells plotted against mVenus, R10 comprises transduced cells<br />
by detecting mVenus expression. <b>E</b>: Overlay of<br />
non-transduced (red) and transduced (green) cells applied against<br />
mVenus.<b>F</b>: Gating non-transduced cells (control) <b>G</b>:<br />
Non-transduced cells applied against mVenus. <b>H</b>:<br />
Gating transduced cells (R2 <span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)<br />
(used plasmids for transfection: GOI: reassembled <b>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(BBa_K404119)</b>, pHelper, pRC). <b>I</b>:<br />
Transduced cells applied against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression. <b>J</b>: Overlay<br />
of non-transduced (red) and transduced (green) cells applied against<br />
mVenus.</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<img id="Diagramm 3"<br />
src="Freiburg10_Modularization_GOI_02_files/image029.gif"<br />
height="355" width="473"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784545">Figure </a>8: Flow cytometry<br />
analysis of vectorplasmids with and without hGH terminator.<span<br />
style="font-weight: normal;"> YFP expression of viral<br />
genomes was determined by flow cytomery after 24-hour post infection.<br />
Results demonstrate that mVenus expression of vectorplasmids lacking<br />
the hGH terminator is reduced significantly proving that the<br />
polyadenylation signal is essential for viral gene expression using<br />
recombinant viral vectors engineered by using components of the Virus<br />
Construction Kit.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoCaption">&nbsp;</p><br />
<h5>Influence of <i>Beta-globin</i> intron Biobrick<br />
on GOI Expression</h5><br />
<p class="MsoNormal">Providing an element assumed to be an<br />
enhancer of transgene<br />
expression (Nott et al. 2003), the iGEM team Freiburg tested a<br />
beta-globin<br />
intron derived from the human <i>beta globin</i> gene<br />
which can be fused<br />
upstream of the desired gene of interest. The beta-globin intron<br />
BioBrick<br />
consists of a partial chimeric CMV promoter followed by the intron II<br />
of the <i>beta-globin</i><br />
gene. The 3´end of the intron is fused to the first 25 bases of human <i>beta<br />
globin</i> gene exon 3. The <i>beta globin</i><br />
intron BioBrick is assumed to<br />
enhance eukaryotic gene expression (Nott et al. 2003). Analysis was<br />
conducted as described for the hGH terminator experiment<br />
(see above). As shown in Figure 9 and Figure 10 the vectorplasmid<br />
missing the <i>beta-globin</i><br />
intron showed a negligible difference in mVenus expression compared to<br />
viral<br />
genomes containing the <i>beta-globin</i> intron.<br />
Considering these results and<br />
taking into account that a constant volume of viral particles has been<br />
used for<br />
transduction, the difference between the construct containing and<br />
lacking the<br />
beta-globin intron is minimal. Since packaging efficiency of the AAV-2<br />
decreases with increasing sizes of the insert (Dong et al. 1996), the<br />
iGEM team Freiburg_Bioware suggests using the <i>beta-globin </i>intron<br />
in dependence on the size of your transgene.</p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="654"><br />
<tbody><br />
<tr style="height: 2.5pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
lacking <i>beta-globin</i> intron</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
id="Grafik 55"<br />
src="Freiburg10_Modularization_GOI_02_files/image030.gif"<br />
height="412" width="640"></p><br />
</td><br />
</tr><br />
<tr style="height: 106.5pt;"><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid<br />
containing <i>beta-globin</i> intron</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
id="Grafik 63"<br />
src="Freiburg10_Modularization_GOI_02_files/image031.gif"<br />
height="410" width="636"></p><br />
</td><br />
</tr><br />
<tr style="height: 106.5pt;"><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"<br />
valign="top" width="654"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784803">Figure </a>9: Flow cytometry<br />
analysis of vectorplasmids with and without <i>beta-globin</i><br />
intron.&nbsp; A<span style="font-weight: normal;">:<br />
Gating non transduced cells (control); subcellular debris and clumps<br />
can be distinguished from single cells by size, estimated forward<br />
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) </span>B<span<br />
style="font-weight: normal;">: Non transduced cells applied<br />
against mVenus (Analytical gate was set such that 1% or fewer of<br />
negative control cells fell within the positive region (R5). </span>C<span<br />
style="font-weight: normal;">: Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: GOI: </span>pSB1C3_lITR_CMV_mVenus_hGH_rITR<br />
(BBa_K404128)<span style="font-weight: normal;">, pHelper,<br />
pRC). </span>D<span style="font-weight: normal;">:<br />
Transduced cells plotted against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression </span>E<span<br />
style="font-weight: normal;">: Overlay of non-transduced<br />
(red) and transduced (green) cells applied against mVenus </span>F<span<br />
style="font-weight: normal;">: Gating non-transduced cells<br />
(control). </span>G<span style="font-weight: normal;">:<br />
Non-transduced cells applied against mVenus (R5).</span>H<span<br />
style="font-weight: normal;">: Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: GOI: reassembled </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(BBa_K404119)<span style="font-weight: normal;">, pHelper,<br />
pRC). </span>I<span style="font-weight: normal;">:<br />
Transduced cells applied against mVenus, R10 comprised transduced<br />
cells, by detecting mVenus expression. </span>J<span<br />
style="font-weight: normal;">: Overlay of non-transduced<br />
(red) and transduced (green) cells applied against mVenus.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="654"><br />
<tbody><br />
<tr style="height: 90.2pt;"><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"<br />
valign="top" width="654"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"<br />
align="left">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><img id="Diagramm 57"<br />
src="Freiburg10_Modularization_GOI_02_files/image032.gif"<br />
height="332" width="450"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784805">Figure </a>10: Flow cytometry<br />
analysis of vectorplasmids with and without <i>beta-globin</i><br />
intron.<span style="font-weight: normal;"> 48-hours post<br />
transfection, viral particles were harvested by freeze-thaw lysis and<br />
centrifugation followed by HT1080 transduction. YFP expression of<br />
vectorplasmids was determined by flow cytometry 24-hours post<br />
infection. The vectorplasmid missing the beta-globin intron showed a<br />
negligible difference in mVenus expression compared to viral plasmid<br />
containing the beta-globin intron.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p><br />
<h5>Functionality of the Full Assembled Vectorplasmid<br />
Demonstrated by GOI Expression<br />
</h5><br />
<p class="MsoNormal">After assembly of plasmids containing<br />
all required elements<br />
(see Figure 1), functionality was tested in cell culture. AAV-293 cells<br />
stably<br />
expressing E1A and E1B proteins were transfected with three plasmids<br />
&nbsp;(pHelper,<br />
pRC, pGOI). Virus particles were harvested 72-hours post-transfection<br />
and the<br />
tumor cell line HT1080 was transduced with the recombinant viral<br />
vectors<br />
encapsidating the gene of interest mVenus (BBa_I757008).</p><br />
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010<br />
compared the standard-plasmid<br />
containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene<br />
system)<br />
with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by<br />
flow<br />
cytometry analysis are shown in Figure 11 and Figure 12. Comparing<br />
mVenus expression<br />
of the standard plasmid and the modified, assembled plasmid reveals<br />
that<br />
biological functionality of the reassembled plasmid was confirmed. </p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span<br />
lang="DE">pSB1C3_mVenus (BBa_K404119)</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img<br />
id="Grafik 2068"<br />
src="Freiburg10_Modularization_GOI_02_files/image033.gif"<br />
height="409" width="634"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pAAV_mVenus<br />
(Stratagene)</b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img<br />
id="Grafik 89"<br />
src="Freiburg10_Modularization_GOI_02_files/image034.gif"<br />
height="406" width="630"></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784576">Figure </a>11: Flow cytometry<br />
analysis of reassembled vectorplasmid (BBa_K404119) compared to<br />
standard plasmid provided by Stratagene. A<span<br />
style="font-weight: normal;">: Gating non transduced cells<br />
(control); subcellular debris and clumps can be distinguished from<br />
single cells by size, estimated forward scatter (FS Lin) and<br />
granularity, estimated side scatter (SS Lin) B: Non transduced cells<br />
plotted against mVenus (Analytical gate was set such that 1% or fewer<br />
of negative control cells fell within the positive region (R5).C:<br />
Gating transduced cells (R2 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R14) (used plasmids for<br />
transfection: pGOI: </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR<br />
(pSB1C3_mVenus: BBa_K404119<span<br />
style="color: rgb(0, 176, 80); font-weight: normal;">)</span><span<br />
style="font-weight: normal;">, pHelper, pRC. </span>D<span<br />
style="font-weight: normal;">: Transduced cells plotted<br />
against mVenus, R10 comprised transduced cells, by detecting mVenus<br />
expression. </span>E<span style="font-weight: normal;">:<br />
Overlay of non-transduced (red) and transduced (green). </span>F<span<br />
style="font-weight: normal;">: Gating non transduced cells<br />
(control). </span>G<span style="font-weight: normal;">:<br />
Non-transduced cells plotted against mVenus (R5). </span>H<span<br />
style="font-weight: normal;">: Gating transduced cells (R14 </span><span<br />
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span<br />
style="font-weight: normal;">R2) (used plasmids for<br />
transfection: pGOI: pAAV_mVenus, pHelper). </span>I<span<br />
style="font-weight: normal;">: Transduced cells plotted<br />
against mVenus, R10 comprised transduced cells, by detecting mVenus<br />
expression.</span> J<span style="font-weight: normal;">:<br />
Overlay of non-transduced (red) and transduced (green) cells plotted<br />
against mVenus expression. </span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p><br />
<table class="MsoTableGrid"<br />
style="border: medium none ; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"<br />
valign="top" width="641"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"<br />
align="center"><img id="Diagramm 90"<br />
src="Freiburg10_Modularization_GOI_02_files/image035.gif"<br />
height="396" width="541"></p><br />
<p class="MsoCaption" style="text-indent: 0cm;"><a<br />
name="_Ref275784852">Figure </a>12: Flow cytometry<br />
analysis of reassembled vectorplasmid (BBa_K404119) compared to<br />
standard plasmid provided by Stratagene. <span<br />
style="font-weight: normal;">Fluorescence of the standard<br />
plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus<br />
(BBa_K404119) construct was measured. As demonstrated mVenus expression<br />
is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the<br />
standard pAAV_mVenus construct.</span></p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
<p class="MsoNormal">&nbsp;</p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Danckwardt, S.,<br />
Hentze, M.W. &amp; Kulozik, A.E., 2008. 3’ end mRNA processing:<br />
molecular<br />
mechanisms and implications for health and disease. <i>The EMBO<br />
journal</i>,<br />
27(3), pp.482-98. Available at:<br />
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Dong, J.Y., Fan,<br />
P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the<br />
packaging<br />
capacity of recombinant adeno-associated virus. <i>Human gene<br />
therapy</i>,<br />
7(17), pp.2101-12. Available at:<br />
http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Millevoi, S. et<br />
al., 2006. An interaction between U2AF 65 and CF I(m) links the<br />
splicing and 3’<br />
end processing machineries. <i>The EMBO journal</i>,<br />
25(20), pp.4854-64.<br />
Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;"><span<br />
style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"<br />
lang="DE">Nott, A., Meislin,<br />
S.H. &amp; Moore, M.J., 2003. A quantitative analysis of intron<br />
effects on<br />
mammalian gene expression. <i>RNA (New York, N.Y.)</i>,<br />
9(5), pp.607-17.<br />
Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p><br />
<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p><br />
</html></div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/SafetyTeam:Freiburg Bioware/Safety2010-10-23T23:00:17Z<p>Kristian: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}} <br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_safety}}<br />
<html><br />
<br><br />
<br />
<h1>Biosafety</h1><br />
<p>Definition: "biosafety measures aim to prevent the unintentional<br />
exposure to pathogens and toxins, or their accidental release" <br />
<a href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br />
</p><br />
<br />
<h2>Risk assessment for our Adeno-associated virus based system</h2><br />
<p><br />
In Germany the "Central Commission for Biological Security" (ZKBS) released three legally binding Risk<br />
Assessment satements for the Adeno-associated Virus <br />
<a href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"></a><sup>26,</sup><br />
<a href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"></a><sup>27,</sup><br />
<a href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"></a><sup>28</sup>.<br />
Risk assessment in other countries may deviate from these decisions, so please inform yourself about the legal regulations on AAV<br />
in your country before using the Virus Construction Kit.<br><br />
</p><br />
<br />
<br />
In Germany the Biological Safety Level(BSL) classifications for AAVs are:<br />
<ul><br />
<li>Adeno-associated Virus 2, 3 and 5 have to be handled <br />
under BSL 1.</li> <br />
<li>Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 have to be <br />
handled under BSL 2.</li> <br />
</ul><br />
<br />
<p>This classification was developed based on the fact that only <br />
serotypes 2, 3 and 5 are isolated from humans and that their harmlessness has <br />
been confirmed in clinical studies. This general classification has to be rechecked when the virus contains gene <br />
sequences with a transforming protential.<br />
</p><br />
<br />
<br />
<p><b>Viral vectors systems</b> packaging a vector plasmid that only contains the<br />
viral Inverted Terminal Repeats (ITRs) and providing the genes for<br />
Rep and Cap in trans (as it is the case for our system) are classified as <b>BSL 1</b> if the following conditions are fullfilled: <br />
<ul><br />
<li>The viral particles do not contain AAV derived sequences other than the ITRs</li> <br />
<li>The viral particles do not contain Nucleotidesequences with a risk potential</li> <br />
</ul><br />
</p><br />
<br />
<p>We also investigated the legal regulations for AAV-2 Viral Vector<br />
systems in the United States. The guidelines of the National Institutes<br />
of Health (NIH) classify in the <br />
<a href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix B</a><sup>32</sup> AAV-2 <br />
Vector Systems as Risk Group 1 (RG1) agents that can be treated under BSL 1. In <br />
detail the appendix states "... adeno- associated virus (AAV) types 1 <br />
through 4; and recombinant AAV constructs, in which the transgene does not <br />
encode either a potentially tumorigenic gene product or a toxin molecule and are <br />
produced in the absence of a helper virus."<br />
</p><br />
<br />
Other general observations with the Adeno-associated Virus 2 AAV-2)are: <br />
<ul><br />
<li>In clinical studies the viral vectors were not <br />
dilivered to the gonades. <br />
<li>Vector sequences were not detectable in the patients blood or<br />
urine at an examination 48 h after an infection with AAV-2<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/10700178"></a><sup>29</sup>. <br />
<li>In absence of Rep proteins, the vector DNA stays extrachromosomal and is not frequently integrated.</li><br />
</ul><br />
<p> <br />
Concluding all these informations and regulations, the project that we have designed in this year <br />
is clearly classified as BLS 1.<br />
</p> <br />
<br />
<h2>General biosafety regulation in Germany</h2><br />
<p><img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" height="auto" width="150"></p><br />
<br />
<br />
<p><br />
</p><br />
<div style="TEXT-ALIGN: justify; MARGIN: 0px 5px; WIDTH: 700px; FLOAT: right; HEIGHT: auto"><span<br />
style="COLOR: darkgreen"><br />
<b>Which specific biosafety rules or guidelines do you have to consider in your country?</b></span><br />
</div><br />
<p></p><br />
<br />
<br><br />
<br />
<p><br />
In Germany all work that includes recombinant DNA technologies is<br />
regulated by the <a href="http://bundesrecht.juris.de/gentg/index.html">Gesetz<br />
zur Regelung der Gentechnik</a>. This law regulates general<br />
aspects in the life sciences and refers for more precise<br />
interpretations in §4 to the <a href="http://bundesrecht.juris.de/gentg/__4.html">Zentrale<br />
Kommission für die Biologische Sicherheit</a>. The ZKBS is a<br />
commission composed of 20 technical experts that releases yearly<br />
statements to actual issues of biosafety. So far the ZKBS released<br />
three stratements affecting the work with Adeno-associated viral<br />
systems<br />
<a href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"><sup>26,</sup></a><br />
<a href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"><sup>27,</sup></a><br />
<a href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"><sup>28</sup></a>.<br />
These documents were used to assess the dangers that could arise from<br />
our project to team members and the enviroment.<br />
</p><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project?</b></span></div><br />
<p><br />
At the Albert-Ludwigs-University Freiburg for all concerns of security the <a href=http://www.sicherheit.uni-freiburg.de>Stabsstelle Sicherheit</a> is responsible and to contact if questions arise. Especially for questions of biological security Dr. Petra Markmeyer-Pieles is cognizant. We contacted her a first time befor the begin of our project in March when it was clear that the Adeno-associated Virus (AAV-2) was chosen as the topic of our project. At that time she proposed to do the cloning in the AAV-2 that is for sure to handle under biological security level 1 and to prepare everything for work under biological security level 2 to satisfy the precaution principle.<br />
The precaution principle was realized and all viral vectors that contained a modified capsid were handled under SII conditions until proven harmless.<br />
In August the planing of the project was completed, summarized in an <a href=https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf>Biosafety application<sup>30</sup></a> and handed to the department for biological security who approve the application in an <a href=https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg>official BSL1 confirmation<sup>31</sup></a>official BSL1 confirmation for our project.</p><br><br><br />
<br />
<br />
<h2>Risk management</h2><br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?</b></span></div><br />
<p><br />
Our project was designed in a way that it avoids any serious safety issues as far as possible.<br />
When working with infectious particles a minimal risk for the researcher is allways present. This risk was minimized by restricting the transduced genes to fluorescent proteins and prodrug convertases that are already proven not to harm human cells in the absece of the corresponding prodrug. A potential danger for the public or the environment was minimized as much as possible by following strictly the rules of Good Laboratory Practice (GLP) and the abdication of using randomized insertions in the capsid and of replication potent viruses. Minimizing the risk for team members and the society was was allways one of the major concerns, especially because worries about undergraduate students manipulating a virus could arise.<br />
The security concept will be explained by quoting and explaining the six guiding principles for safe manipulation of Gene Manipulated Organisms (GMOs) as summarized in Kimman et al. ; 2008<a href=http://www.ncbi.nlm.nih.gov/pubmed/18625678><sup>18</sup></a>.<br />
<li><b>1) Hazard recognition and identification</b><br />
Risk assessment has been done and all legal regulations were considered as described in the last paragraph.</li><br />
<li><b>2) Biological containment</b><br />
Biological containment means the usage of organisms with "reduced replicative capacity, inefectivity , transmissibility, and virulence"18.<br />
For our project only replicative deficient viruses were used, additional all modifications aimed to have a specific targeting of the resulting viral vectors for a specific cell type. This gain in specificity requires it to cut off the braod natural tropism resulting is an less infective virus copared to the wild type virus.</li><br />
<li><b>3) Concentration and enclosure</b><br />
All working steps for the preparation of viral vectors were carried out in a BSL II laboratory and within this laboratory work with the AAV was restricted to a separate Laminar flow cabinet type II. Cell culture and storage of the virus was also done in separate Freezers and incubators. All laboratories and epipment that contained viral vectors were specially marked, for example with a Biohazard warning signs.</li><br />
<li><b>4) Exposure minimization</b><br />
This aspect of the guiding principles can be sumarized under "operator protection"18. The Exposure minimization was achieved in our laboratory by wearing special labcoats for the SII laboratory and gloves that were desinfected and changed regualrely. During manipulation of viral vectors attention was payed to avoid droplets and especially aerosoles. A possible diversion of the viral vectors was avoided by cleaning all equipment when inserted or removed from the Laminar flow cabinet and after completion of the work task.</li><br />
<li><b>5) Physical containment</b><br />
The requirements for the physical containment were fullfilled by performing all manipulation on the AAV in an BSL II laboratory that guaranted a restriction of persons that entered the laboratory.</li><br />
<li><b>6) Hazard minimization</b><br />
For the AAV-2 there are no sugestive activitis because the possible danger that runs out of the AAV is comparably low, vaccination is not avilible and biomonitoring is not necessary.</li></p><br><br><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?</b></span></div><br />
<p><br />
<img align="right" width="300px" height="auto" margin: 0px 5px 0px 5px src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_Warning_SignI.png" alt="Warning sign for part descriptions" /><br />
Several composite parts that were assembled by our Team this year are alone capable of producing infectious viral particles when transduced together with a vector plasmid and a helper plasmid into <a href=https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf>AAV-293</a> cells. These special cells provide the adenoviral gene E1 stabily integrated in trans.<br />
These cells are not provided in the Virus Construction Kit nor availible in the Parts Registry and have to purchased from other laboratories or a commercial supplyer.<br />
For this reason we estimate the risk of a accidental transformation of <a href=https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf>AAV-293</a> cells with all three plasmids for negligible.<br />
Nevertheless we considered it useful to mark every BioBrick or Composite Part in the Registry that contributes to the production or is capable of producing viral vectors when transformed under the previously mentioned conditions.<br />
</p><br><br><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Contribute to community discussions on what needs to go into a code against the use of our science for hostile purposes (see A Community Response)</b></span></div><br />
<p><br />
<li>Public Perception<br />
<li>importance of maintaining public legitimacy and support</li><br />
<li>apllications should demonstrate clear social benefits</li><br />
<li>not overhyped - anxiety and unrealistic hopes</li><br />
<br />
<blockquote>psychological research into the concept of "identity-driven decision-making" (Torpman,2004)<a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452> <sup>19</sup></a></blockquote><br />
<br />
<blockquote>Every grout has a set of norms: a code of conduct about what is acceptable beahviour (Jaques, 2004]<a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452> <sup>19</sup></a></blockquote><br />
</p><br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center><h2>Trade-off between potential misuse and promising medical progress</h2></center><br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</b> </span></div><br />
<p><br />
In principle each research-project that bears any risks for engaged researchers, mankind or the environment should be treated under the precautionary principle as proposed <a href=http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf><sup>11</sup></a>: "treat synthetic microorganisms as dangerous until proven harmless".<br><br />
This would mean to work on such synthetic DNA containing Bio Bricks at least under Biological security levels two.<br><br />
Additional to this secure working environment the system itself can be optimized according to biosafety aspects, means to reduce it's viability outside the laboratory. This aim can be approached by reducing the systems ability to evolve, proliferate and interact with it's environment. A common method to achieve this goal is to engineer microorganisms in a way that they depend on nutrients that can't be found in the environment in sufficient amount.<br />
<br />
</p><br><br><br />
<br />
<br />
<center><h1>Biosecurity</h1></center><br />
Def: "measures focus on the prevention of theft, misuse , or intentional relese of pathogens and toxins" <a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf><sup>1</sup></a><br><br />
<br />
<br />
<center><h2>The malignant use of biological agents in history</h2></center><br />
<p>The misuse of biological agent as weapons in warfare is a fear spreading companion in the history of mankind, ranging from the well-poisoners in prehistoric times to bio-terrorists present days. The following brakt intends to give a short outline of the major events<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a>:<br />
<br />
<li> First systhematic use of chemical weapons during the first world war</li><br />
<li> The 1918 flu pandemic lasted from March 1918 to June 1920 and killed approximately 50 Million people around the world. Rumors circulated that this pandemic is caused by the other combatant nation.</li><br />
<li> "Prohibion of the Use of Asphyxiating, Poisonous or other Gases and of Bacteriological Methods of Warfare" was signed on 19 June 1925<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a></li><br />
<li> Several combatant nations of the second world war established biological warfare programs. </li><br />
<li> The range of the Japanese biological warfare program lead several nations to expand their own biological warfare program.</li><br />
<li> Limited military use<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a> and the advances in molecular biology (e.g. the first isolatin of a gene and the discovery of the restriction enzymes in 1969) lead to the "Convention on the Prohibion of the Development, Production and Stockpilling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction " (BTWC) on 10 April 1972. <br />
<blockquote>''"develop, produce, stockpile or otherwise acquire or retain: ... Microbial or other biological agents, or toxins whatever their origin or method of production, of types and in wantities that have no justification for prophylactic, protective or other peaceful purpose..." ''<sup>3</sup></blockquote></li><br />
<li> Breach of the BTWC by the Soviet Union which continued their offensive biologicla warfare programm<br />
<li> Stop of the biological warfare Program "Biopreparat" accompanies the Dissolution of the Svoviet Union in 1991</li><br />
<li> Antrax attacks in the USA in 2001</li><br />
<br />
After this short description of misused biological components there is still the question what impact biological warfare will have in the future of mankind.<br />
</p><br />
<br />
<center><h2>Broad avilibility of knowledge</h2></center><br />
n the life sciences information has allways been freely acessible for everybody who is interested in the results of a particular research project. The combination of this global availibility with the new possibilities of the internet broad for literature search and availibility of gene sequences made it easiere to collect informations for a possible misuse. <br><br />
For the overwhelming majority this open availibility is absolutely desirely but on the other hand there are also examples of research results that bear a very high risk to be misused. <br><br />
Following we present the three most controversial discussed publications that could also be read as a "How to create your own bioweapon".<br />
<br />
<br />
<h3>Mousepox Virus in Australien</h3><br><br />
The Australian research group around <a href=http://www.ncbi.nlm.nih.gov/pubmed/11152493> Jackson et al. ; 2001 <sup>16</sup></a>inserted the coding region of the IL-4 gne into the genome of mousepox. They hoped to create a virus that sterilizes mice and thus provides a means for pest control. Contrarely to their expectations they had created a superstrain that killed even naturally resistent mice and mice that had been vaccinated against normal mousepox. This discovery could potentially be used to make smallpox resistant to potential vaccines.<br />
<br />
<h3>Synthesis of a Polio Virus</h3><br><br />
At the State University of New York <a href=http://www.sciencemag.org/cgi/content/full/297/5583/1016> Cello et al. ; 2002 <sup>05</sup></li> synthesized a "living" polio virus from scratch. Especially the <a href=http://www.sciencemag.org/cgi/content/full/1072266/DC1>Supporting Online Material</a> caused worried remarks because it precisely describes how to assemble a virus from small oligo nucleotides. The authors jusifyed their project by declaring that they:<br />
<blockquote>"made the virus to send a warning that terrorists might be able to make biological weapons without obtaining a natrual virus"<a href=http://www.ncbi.nlm.nih.gov/pubmed/19784453><sup>17</sup></a></blockquote><br />
<br />
<h3>Reconstitution of the Spanish Flu</h3><br><br />
<img width="250px" class="right" src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Spanishflu.jpg" alt="Influenza hospital for american soldiers" /><br />
At the Center for Disease Control and Prevention (CDC) the group around <a href=http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf>Tumpey et al. ; 2005<sup>24</sup></a> published that they had sequenced and recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50 million people. The publication of the sequence provoked several very different responses that can only be partially be quoted here.<br />
<br />
The controversial noticed outrider of the Synthetic Biology Craig Venter seen in the new virus<br />
<blockquote>"the first true Juressic Parc scenario" <a href=http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&sID=4233><sup>2</sup></a></li></blockquote><br />
<br />
The Institute Professor at the MIT Philip A. Sharp supported the publication because he:<br />
<blockquote>"belive[s] that allowing the publication of this information was the correct decision in terms of both national security and public health" <a href=http://www.sciencemag.org/cgi/content/short/310/5745/17><sup>12</sup></a></blockquote><br />
<br />
Wheras <a href=http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf>v. Bubnoff; 2005<sup>25</sup></a> critisizes the CDS for it's careless regulations for the shipment of viruses and the willingness to propagate highly virulent viruses. This causes in his opinion the risk of possible accidents resulting in the release of the virus to the environment.<br />
<br />
<br />
<center><h2>Broad avilibility of material</h2></center><br />
<br />
<center><h2>Szenarios of attack</h2></center><br />
<br />
<center><h2>Conclusion</h2></center><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Include something in your project description and presentations that demonstrates that you have thought about how others could misuse your work.</b> </span></div><br />
<p><br />
For sure there is allway the possibility that knowledge to produce transgene viral vectors could be used to produce bioweapons. Therefor it was important for us to use a system that does not bear the risk that someone could use it for evil purpose. In the case of the Adeno-associated virus the very limited packaging capacity is the major reason that excludes it from the list of agents that could realistically be used for the pruduction of bioweapons. Even a fully replication potent AAV will depend on the coninfection of a helpervirus and is therefore not suitable for a fast propagation in an population.<br />
Additional to this point we concentrated our project on the retargeting of the virus - means to make the broad tropismn more narrow and to decrease the transduction efficiency in the most cases. This modification is usually mainly required for medical purposes. Also we did neither investigate possibilities to shield the vector from the immune system of potential host nor ways to bypass an existing immunity. <br />
</p><br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<li>01 <a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf>Synthetic Biology & Biosecurity - Awareness in Europe | Kelle ; 2007<sup>1</sup></a></li><br />
<br />
<li>02 <a href=http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&sID=4233>Synthetic Biology - Social and Ethical Challenges | Balmer et Martin ; 2008<sup>2</sup></a></li><br />
<br />
<li>03) Synthetic Biology - Applying Engineering to Biology<br />
<br> used<br />
<br />
<li>04 <a href=http://www.informaworld.com/smpp/content~content=a713604665&db=all>The Impact of the Development of Modern Biology and Medicine on the Evolution of Offensive Biological Warfare Programs in the Twentieth Century | Dando ; 1999 <sup>04</sup></a></li><br />
<br />
<li>05 <a href=http://www.sciencemag.org/cgi/content/full/297/5583/1016>Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template | Cello et al. ; 2002 <sup>05</sup></li><br />
<br />
<li>06 <a href=http://www.nytimes.com/2003/12/03/opinion/03iht-edstein_ed3_.html>Dangerous research : When science breeds nightmares | Steinbruner et Harris ; 2003 <sup>06</sup></a></li><br />
<br />
<li>07 <a href=http://www.jcvi.org/cms/fileadmin/site/research/projects/synthetic-genomics-report/synthetic-genomics-report.pdf>Synthetic Genomics - Options for governance | Garfinkel et al. ; 2007 <sup>07</sup></a></li><br />
<br />
<li>08 <a href=http://www.nature.com/nrg/journal/v6/n7/abs/nrg1637.html>Synthetic biology | Benner et Sismour , 2005 <sup>08</sup></a></li><br />
<br />
<li>09 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16954140>Synthetic biology—putting engineering into biology | Heinemann et Panke ; 2006 <sup>09</sup></a></li><br />
<br />
<li>10 <a href=http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3>Revealed: the lax laws that could allow assembly of deadly virus DNA | Randerson ; 2006 <sup>10</sup></a> </li><br />
<br />
<li>11 <a href=http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf>The Promise and Perils of Synthetic Biology | Tucker & Zilinskas ; 2006 <sup>11</sup></a></li><br />
<br />
<li>12 <a href=http://www.sciencemag.org/cgi/content/short/310/5745/17>1918 Flu and Responsible Science | Sharp ; 2005 <sup>12</sup></a></li><br />
<br />
<li>13 <a href=http://www.fas.org/irp/cia/product/bw1103.pdf> The Darker Bioweapons Future | CIA ; 2003 <sup>13</sup></a></li><br />
<br />
<li>14) [[Media:Primer for Synthetic Biology - Mohr 2007.pdf]]<br />
</li><br />
<br />
<li>15) [[Media:Freiburg10_The bugs of war.pdf]]<br />
</li><br />
<br />
<li>16 <a href=http://www.ncbi.nlm.nih.gov/pubmed/11152493>Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. | Jackson et al. ; 2001 <sup>16</sup></a></li><br />
<br />
<li>17 <a href=http://www.ncbi.nlm.nih.gov/pubmed/19784453>Governance of dual-use research: an ethical dilemma. | Selgelid ; 2009 <sup>17</sup></a></li><br />
<br />
<li>18 <a href=http://www.ncbi.nlm.nih.gov/pubmed/18625678>Evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures. | Kimman et al. 2008 <sup>18</sup></a> </li><br />
<br />
<li>19) <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452>A Hippocratic Oath for life scientists | Revill et Dando ; 2006 <sup>19</sup></a></li><br />
<br />
<li>20 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819443>Empowerment and restraint in scientific communication. New developments make it easier to share information, but more difficult to deal with dual-use biology. | Campbell ; 2006<sup>20</sup></a></li><br />
<br />
<li>21 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819441>When risk outweighs benefit | Aken ; 2006 <sup>21</sup></a></li><br />
<br />
<li>22 <a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408>Advances in life sciences and bioterrorism. Risks, perspectives and responsibilities.| Beck ; 2003 <sup>22</sup></a></li><br />
<br />
<li>23 <a href=http://www.ncbi.nlm.nih.gov/pubmed/12590130>PNAS policy on publication of sensitive material in the life sciences | Cozzarelli ; 2003 <sup>23</sup></a></li><br />
<br />
<li>24 <a href=http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf>Characterization of the<br />
Reconstructed 1918 Spanish<br />
Influenza Pandemic Virus | Tumpey et al. ; 2005<sup>24</sup></a> </li><br />
<br />
<li>25 <a href=http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf>Deadly flu virus can be<br />
sent through the mail| v. Bubnoff; 2005<sup>25</sup></a></li><br />
<br />
<li>26 <a href=https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf>Risk assessment of human Adeno-associated viruses| ZKBS; 2001<sup>26</sup></a></li><br />
<br />
<li>27 <a href=https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf>Advises for AAV carrying cell cycle regulating genes| ZKBS; 2004 <sup>27</sup></a></li><br />
<br />
<li>28 <a href=https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf>Risk assessment of human Adeno-associated viruses and AAV derived vectors| ZKBS; 2005 <sup>28</sup></a> </li><br />
<br />
<li>29 <a href=http://www.ncbi.nlm.nih.gov/pubmed/10700178>Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.| Kai et al. ; 2000 <sup>29</sup></a><br />
Bioverteilung in Klinischer Studie </li><br />
<br />
<li>30 <a href=https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf>Biosafety application of the iGEM team Freiburg_Bioware 2010<sup>30</sup></a> (in German)</li><br />
<br />
<li>31 <a href=https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg>Official classification as Biological Safety Level 1 by the local biosafety office<sup>31</sup></a></li><br />
<br />
<li>32 <a href=http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm>Appendix B | National Institute of Health <sup>32</sup></a></li><br />
<br />
</html></div>Kristianhttp://2010.igem.org/Team:Freiburg_Bioware/SafetyTeam:Freiburg Bioware/Safety2010-10-23T22:50:55Z<p>Kristian: </p>
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<br />
<h1>Biosafety</h1><br />
<p>Definition: "biosafety measures aim to prevent the unintentional<br />
exposure to pathogens and toxins, or their accidental release" <br />
<a href="http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf"><sup>1</sup></a><br />
</p><br />
<br />
<h2>Risk assessment for our Adeno-associated virus based system</h2><br />
<p><br />
In Germany the "Central Commission for Biological Security" (ZKBS) released three legally binding Risk<br />
Assessment satements for the Adeno-associated Virus <br />
<a href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"></a><sup>26,</sup><br />
<a href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"></a><sup>27,</sup><br />
<a href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"></a><sup>28</sup>.<br />
Risk assessment in other countries may deviate from these decisions, so please inform yourself about the legal regulations on AAV<br />
in your country before using the Virus Construction Kit.<br><br />
</p><br />
<br />
<br />
In Germany the classification in Biological Safety Level (BSL) is:<br />
<ul><br />
<li>Adeno-associated Virus 2, 3 and 5 are to handle <br />
under BSL&nbsp;1&nbsp; <br />
<li>Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 are <br />
to handle under BSL 2. This classification was developed based on the fact that only <br />
serotypes 2, 3 and 5 are isolated from humans and that their harmlessness has <br />
been confirmed in clinical studies.</li> <br />
</ul><br />
<br />
<p>This general classification has to be rechecked when the virus contains gene <br />
sequences with a transforming protential.<br />
</p><br />
<br />
<p><br />
Viral vectors systems packaging a vector plasmid that only contains the<br />
viral Inverted Terminal Repeats (ITRs) and providing the genes for<br />
Rep and Cap in trans (as it is the case for our system) are classified as <b>BSL 1</b> if the following conditions are fullfilled: <br />
</p><br />
<ul><br />
<li>The viral particles do not contain AAV derived <br />
sequences other than the ITRs <br />
<li>The viral particles do not contain Nucleotidesequences with a risk potential</li> <br />
</ul><br />
<br />
<br />
<p>We also investigated the legal regulations for AAV-2 Viral Vector<br />
systems in the United States. The guidelines of the National Institutes<br />
of Health (NIH) classify in the <br />
<a href="http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm">Appendix B</a><sup>32</sup> AAV-2 <br />
Vector Systems as Risk Group 1 (RG1) agents that can be treated under BSL 1. In <br />
detail the appendix states "... adeno- associated virus (AAV) types 1 <br />
through 4; and recombinant AAV constructs, in which the transgene does not <br />
encode either a potentially tumorigenic gene product or a toxin molecule and are <br />
produced in the absence of a helper virus."<br />
</p><br />
<br />
General observations with the Adeno-associated Virus 2 AAV-2): <br />
<ul><br />
<li>In clinical studies the viral vectors were not <br />
dilivered to the gonades. <br />
<li>Vector sequences were not detectable in the patients blood or<br />
urine at an examination 48 h after an infection with AAV-2<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/10700178"></a><sup>29</sup>. <br />
<li>In absece of Rep proteins, the vector DNA stays extrachromosomal and is not frequently integrated.</li><br />
</ul><br />
<p> <br />
Concluding all these informations and regulations, the project that we have designed in this year <br />
is clearly classified as BLS 1.<br />
</p> <br />
<br />
<h2>General biosafety regulation in Germany</h2><br />
<p><img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" height="auto" width="150"></p><br />
<br />
<br />
<p><br />
</p><br />
<div style="TEXT-ALIGN: justify; MARGIN: 0px 5px; WIDTH: 700px; FLOAT: right; HEIGHT: auto"><span<br />
style="COLOR: darkgreen"><br />
<b>Which specific biosafety rules or guidelines do you have to consider in your country?</b></span><br />
</div><br />
<p></p><br />
<br />
<br><br />
<br />
<p><br />
In Germany all work that includes recombinant DNA technologies is<br />
regulated by the <a href="http://bundesrecht.juris.de/gentg/index.html">Gesetz<br />
zur Regelung der Gentechnik</a>. This law regulates general<br />
aspects in the life sciences and refers for more precise<br />
interpretations in §4 to the <a href="http://bundesrecht.juris.de/gentg/__4.html">Zentrale<br />
Kommission für die Biologische Sicherheit</a>. The ZKBS is a<br />
commission composed of 20 technical experts that releases yearly<br />
statements to actual issues of biosafety. So far the ZKBS released<br />
three stratements affecting the work with Adeno-associated viral<br />
systems<br />
<a href="https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf"><sup>26,</sup></a><br />
<a href="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf"><sup>27,</sup></a><br />
<a href="https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf"><sup>28</sup></a>.<br />
These documents were used to assess the dangers that could arise from<br />
our project to team members and the enviroment.<br />
</p><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project?</b></span></div><br />
<p><br />
At the Albert-Ludwigs-University Freiburg for all concerns of security the <a href=http://www.sicherheit.uni-freiburg.de>Stabsstelle Sicherheit</a> is responsible and to contact if questions arise. Especially for questions of biological security Dr. Petra Markmeyer-Pieles is cognizant. We contacted her a first time befor the begin of our project in March when it was clear that the Adeno-associated Virus (AAV-2) was chosen as the topic of our project. At that time she proposed to do the cloning in the AAV-2 that is for sure to handle under biological security level 1 and to prepare everything for work under biological security level 2 to satisfy the precaution principle.<br />
The precaution principle was realized and all viral vectors that contained a modified capsid were handled under SII conditions until proven harmless.<br />
In August the planing of the project was completed, summarized in an <a href=https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf>Biosafety application<sup>30</sup></a> and handed to the department for biological security who approve the application in an <a href=https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg>official BSL1 confirmation<sup>31</sup></a>official BSL1 confirmation for our project.</p><br><br><br />
<br />
<br />
<h2>Risk management</h2><br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?</b></span></div><br />
<p><br />
Our project was designed in a way that it avoids any serious safety issues as far as possible.<br />
When working with infectious particles a minimal risk for the researcher is allways present. This risk was minimized by restricting the transduced genes to fluorescent proteins and prodrug convertases that are already proven not to harm human cells in the absece of the corresponding prodrug. A potential danger for the public or the environment was minimized as much as possible by following strictly the rules of Good Laboratory Practice (GLP) and the abdication of using randomized insertions in the capsid and of replication potent viruses. Minimizing the risk for team members and the society was was allways one of the major concerns, especially because worries about undergraduate students manipulating a virus could arise.<br />
The security concept will be explained by quoting and explaining the six guiding principles for safe manipulation of Gene Manipulated Organisms (GMOs) as summarized in Kimman et al. ; 2008<a href=http://www.ncbi.nlm.nih.gov/pubmed/18625678><sup>18</sup></a>.<br />
<li><b>1) Hazard recognition and identification</b><br />
Risk assessment has been done and all legal regulations were considered as described in the last paragraph.</li><br />
<li><b>2) Biological containment</b><br />
Biological containment means the usage of organisms with "reduced replicative capacity, inefectivity , transmissibility, and virulence"18.<br />
For our project only replicative deficient viruses were used, additional all modifications aimed to have a specific targeting of the resulting viral vectors for a specific cell type. This gain in specificity requires it to cut off the braod natural tropism resulting is an less infective virus copared to the wild type virus.</li><br />
<li><b>3) Concentration and enclosure</b><br />
All working steps for the preparation of viral vectors were carried out in a BSL II laboratory and within this laboratory work with the AAV was restricted to a separate Laminar flow cabinet type II. Cell culture and storage of the virus was also done in separate Freezers and incubators. All laboratories and epipment that contained viral vectors were specially marked, for example with a Biohazard warning signs.</li><br />
<li><b>4) Exposure minimization</b><br />
This aspect of the guiding principles can be sumarized under "operator protection"18. The Exposure minimization was achieved in our laboratory by wearing special labcoats for the SII laboratory and gloves that were desinfected and changed regualrely. During manipulation of viral vectors attention was payed to avoid droplets and especially aerosoles. A possible diversion of the viral vectors was avoided by cleaning all equipment when inserted or removed from the Laminar flow cabinet and after completion of the work task.</li><br />
<li><b>5) Physical containment</b><br />
The requirements for the physical containment were fullfilled by performing all manipulation on the AAV in an BSL II laboratory that guaranted a restriction of persons that entered the laboratory.</li><br />
<li><b>6) Hazard minimization</b><br />
For the AAV-2 there are no sugestive activitis because the possible danger that runs out of the AAV is comparably low, vaccination is not avilible and biomonitoring is not necessary.</li></p><br><br><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?</b></span></div><br />
<p><br />
<img align="right" width="300px" height="auto" margin: 0px 5px 0px 5px src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_Warning_SignI.png" alt="Warning sign for part descriptions" /><br />
Several composite parts that were assembled by our Team this year are alone capable of producing infectious viral particles when transduced together with a vector plasmid and a helper plasmid into <a href=https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf>AAV-293</a> cells. These special cells provide the adenoviral gene E1 stabily integrated in trans.<br />
These cells are not provided in the Virus Construction Kit nor availible in the Parts Registry and have to purchased from other laboratories or a commercial supplyer.<br />
For this reason we estimate the risk of a accidental transformation of <a href=https://static.igem.org/mediawiki/2010/e/e0/Freiburg10_AAv293_cell_line.pdf>AAV-293</a> cells with all three plasmids for negligible.<br />
Nevertheless we considered it useful to mark every BioBrick or Composite Part in the Registry that contributes to the production or is capable of producing viral vectors when transformed under the previously mentioned conditions.<br />
</p><br><br><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Contribute to community discussions on what needs to go into a code against the use of our science for hostile purposes (see A Community Response)</b></span></div><br />
<p><br />
<li>Public Perception<br />
<li>importance of maintaining public legitimacy and support</li><br />
<li>apllications should demonstrate clear social benefits</li><br />
<li>not overhyped - anxiety and unrealistic hopes</li><br />
<br />
<blockquote>psychological research into the concept of "identity-driven decision-making" (Torpman,2004)<a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452> <sup>19</sup></a></blockquote><br />
<br />
<blockquote>Every grout has a set of norms: a code of conduct about what is acceptable beahviour (Jaques, 2004]<a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452> <sup>19</sup></a></blockquote><br />
</p><br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center><h2>Trade-off between potential misuse and promising medical progress</h2></center><br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</b> </span></div><br />
<p><br />
In principle each research-project that bears any risks for engaged researchers, mankind or the environment should be treated under the precautionary principle as proposed <a href=http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf><sup>11</sup></a>: "treat synthetic microorganisms as dangerous until proven harmless".<br><br />
This would mean to work on such synthetic DNA containing Bio Bricks at least under Biological security levels two.<br><br />
Additional to this secure working environment the system itself can be optimized according to biosafety aspects, means to reduce it's viability outside the laboratory. This aim can be approached by reducing the systems ability to evolve, proliferate and interact with it's environment. A common method to achieve this goal is to engineer microorganisms in a way that they depend on nutrients that can't be found in the environment in sufficient amount.<br />
<br />
</p><br><br><br />
<br />
<br />
<center><h1>Biosecurity</h1></center><br />
Def: "measures focus on the prevention of theft, misuse , or intentional relese of pathogens and toxins" <a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf><sup>1</sup></a><br><br />
<br />
<br />
<center><h2>The malignant use of biological agents in history</h2></center><br />
<p>The misuse of biological agent as weapons in warfare is a fear spreading companion in the history of mankind, ranging from the well-poisoners in prehistoric times to bio-terrorists present days. The following brakt intends to give a short outline of the major events<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a>:<br />
<br />
<li> First systhematic use of chemical weapons during the first world war</li><br />
<li> The 1918 flu pandemic lasted from March 1918 to June 1920 and killed approximately 50 Million people around the world. Rumors circulated that this pandemic is caused by the other combatant nation.</li><br />
<li> "Prohibion of the Use of Asphyxiating, Poisonous or other Gases and of Bacteriological Methods of Warfare" was signed on 19 June 1925<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a></li><br />
<li> Several combatant nations of the second world war established biological warfare programs. </li><br />
<li> The range of the Japanese biological warfare program lead several nations to expand their own biological warfare program.</li><br />
<li> Limited military use<a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408><sup>22</sup></a> and the advances in molecular biology (e.g. the first isolatin of a gene and the discovery of the restriction enzymes in 1969) lead to the "Convention on the Prohibion of the Development, Production and Stockpilling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction " (BTWC) on 10 April 1972. <br />
<blockquote>''"develop, produce, stockpile or otherwise acquire or retain: ... Microbial or other biological agents, or toxins whatever their origin or method of production, of types and in wantities that have no justification for prophylactic, protective or other peaceful purpose..." ''<sup>3</sup></blockquote></li><br />
<li> Breach of the BTWC by the Soviet Union which continued their offensive biologicla warfare programm<br />
<li> Stop of the biological warfare Program "Biopreparat" accompanies the Dissolution of the Svoviet Union in 1991</li><br />
<li> Antrax attacks in the USA in 2001</li><br />
<br />
After this short description of misused biological components there is still the question what impact biological warfare will have in the future of mankind.<br />
</p><br />
<br />
<center><h2>Broad avilibility of knowledge</h2></center><br />
n the life sciences information has allways been freely acessible for everybody who is interested in the results of a particular research project. The combination of this global availibility with the new possibilities of the internet broad for literature search and availibility of gene sequences made it easiere to collect informations for a possible misuse. <br><br />
For the overwhelming majority this open availibility is absolutely desirely but on the other hand there are also examples of research results that bear a very high risk to be misused. <br><br />
Following we present the three most controversial discussed publications that could also be read as a "How to create your own bioweapon".<br />
<br />
<br />
<h3>Mousepox Virus in Australien</h3><br><br />
The Australian research group around <a href=http://www.ncbi.nlm.nih.gov/pubmed/11152493> Jackson et al. ; 2001 <sup>16</sup></a>inserted the coding region of the IL-4 gne into the genome of mousepox. They hoped to create a virus that sterilizes mice and thus provides a means for pest control. Contrarely to their expectations they had created a superstrain that killed even naturally resistent mice and mice that had been vaccinated against normal mousepox. This discovery could potentially be used to make smallpox resistant to potential vaccines.<br />
<br />
<h3>Synthesis of a Polio Virus</h3><br><br />
At the State University of New York <a href=http://www.sciencemag.org/cgi/content/full/297/5583/1016> Cello et al. ; 2002 <sup>05</sup></li> synthesized a "living" polio virus from scratch. Especially the <a href=http://www.sciencemag.org/cgi/content/full/1072266/DC1>Supporting Online Material</a> caused worried remarks because it precisely describes how to assemble a virus from small oligo nucleotides. The authors jusifyed their project by declaring that they:<br />
<blockquote>"made the virus to send a warning that terrorists might be able to make biological weapons without obtaining a natrual virus"<a href=http://www.ncbi.nlm.nih.gov/pubmed/19784453><sup>17</sup></a></blockquote><br />
<br />
<h3>Reconstitution of the Spanish Flu</h3><br><br />
<img width="250px" class="right" src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Spanishflu.jpg" alt="Influenza hospital for american soldiers" /><br />
At the Center for Disease Control and Prevention (CDC) the group around <a href=http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf>Tumpey et al. ; 2005<sup>24</sup></a> published that they had sequenced and recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50 million people. The publication of the sequence provoked several very different responses that can only be partially be quoted here.<br />
<br />
The controversial noticed outrider of the Synthetic Biology Craig Venter seen in the new virus<br />
<blockquote>"the first true Juressic Parc scenario" <a href=http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&sID=4233><sup>2</sup></a></li></blockquote><br />
<br />
The Institute Professor at the MIT Philip A. Sharp supported the publication because he:<br />
<blockquote>"belive[s] that allowing the publication of this information was the correct decision in terms of both national security and public health" <a href=http://www.sciencemag.org/cgi/content/short/310/5745/17><sup>12</sup></a></blockquote><br />
<br />
Wheras <a href=http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf>v. Bubnoff; 2005<sup>25</sup></a> critisizes the CDS for it's careless regulations for the shipment of viruses and the willingness to propagate highly virulent viruses. This causes in his opinion the risk of possible accidents resulting in the release of the virus to the environment.<br />
<br />
<br />
<center><h2>Broad avilibility of material</h2></center><br />
<br />
<center><h2>Szenarios of attack</h2></center><br />
<br />
<center><h2>Conclusion</h2></center><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_iGEMs_question.png" width="150" <br />
height="auto"/><br />
<div style="float:right; width:700px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"><br />
<span style="color:DarkGreen"><b>Include something in your project description and presentations that demonstrates that you have thought about how others could misuse your work.</b> </span></div><br />
<p><br />
For sure there is allway the possibility that knowledge to produce transgene viral vectors could be used to produce bioweapons. Therefor it was important for us to use a system that does not bear the risk that someone could use it for evil purpose. In the case of the Adeno-associated virus the very limited packaging capacity is the major reason that excludes it from the list of agents that could realistically be used for the pruduction of bioweapons. Even a fully replication potent AAV will depend on the coninfection of a helpervirus and is therefore not suitable for a fast propagation in an population.<br />
Additional to this point we concentrated our project on the retargeting of the virus - means to make the broad tropismn more narrow and to decrease the transduction efficiency in the most cases. This modification is usually mainly required for medical purposes. Also we did neither investigate possibilities to shield the vector from the immune system of potential host nor ways to bypass an existing immunity. <br />
</p><br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<li>01 <a href=http://www.idialog.eu/uploads/file/Synbiosafe-Biosecurity_awareness_in_Europe_Kelle.pdf>Synthetic Biology & Biosecurity - Awareness in Europe | Kelle ; 2007<sup>1</sup></a></li><br />
<br />
<li>02 <a href=http://www.bbsrc.ac.uk/nmsruntime/saveasdialog.aspx?lID=2277&sID=4233>Synthetic Biology - Social and Ethical Challenges | Balmer et Martin ; 2008<sup>2</sup></a></li><br />
<br />
<li>03) Synthetic Biology - Applying Engineering to Biology<br />
<br> used<br />
<br />
<li>04 <a href=http://www.informaworld.com/smpp/content~content=a713604665&db=all>The Impact of the Development of Modern Biology and Medicine on the Evolution of Offensive Biological Warfare Programs in the Twentieth Century | Dando ; 1999 <sup>04</sup></a></li><br />
<br />
<li>05 <a href=http://www.sciencemag.org/cgi/content/full/297/5583/1016>Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template | Cello et al. ; 2002 <sup>05</sup></li><br />
<br />
<li>06 <a href=http://www.nytimes.com/2003/12/03/opinion/03iht-edstein_ed3_.html>Dangerous research : When science breeds nightmares | Steinbruner et Harris ; 2003 <sup>06</sup></a></li><br />
<br />
<li>07 <a href=http://www.jcvi.org/cms/fileadmin/site/research/projects/synthetic-genomics-report/synthetic-genomics-report.pdf>Synthetic Genomics - Options for governance | Garfinkel et al. ; 2007 <sup>07</sup></a></li><br />
<br />
<li>08 <a href=http://www.nature.com/nrg/journal/v6/n7/abs/nrg1637.html>Synthetic biology | Benner et Sismour , 2005 <sup>08</sup></a></li><br />
<br />
<li>09 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16954140>Synthetic biology—putting engineering into biology | Heinemann et Panke ; 2006 <sup>09</sup></a></li><br />
<br />
<li>10 <a href=http://www.guardian.co.uk/world/2006/jun/14/terrorism.topstories3>Revealed: the lax laws that could allow assembly of deadly virus DNA | Randerson ; 2006 <sup>10</sup></a> </li><br />
<br />
<li>11 <a href=http://www.thenewatlantis.com/docLib/TNA12-TuckerZilinskas.pdf>The Promise and Perils of Synthetic Biology | Tucker & Zilinskas ; 2006 <sup>11</sup></a></li><br />
<br />
<li>12 <a href=http://www.sciencemag.org/cgi/content/short/310/5745/17>1918 Flu and Responsible Science | Sharp ; 2005 <sup>12</sup></a></li><br />
<br />
<li>13 <a href=http://www.fas.org/irp/cia/product/bw1103.pdf> The Darker Bioweapons Future | CIA ; 2003 <sup>13</sup></a></li><br />
<br />
<li>14) [[Media:Primer for Synthetic Biology - Mohr 2007.pdf]]<br />
</li><br />
<br />
<li>15) [[Media:Freiburg10_The bugs of war.pdf]]<br />
</li><br />
<br />
<li>16 <a href=http://www.ncbi.nlm.nih.gov/pubmed/11152493>Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. | Jackson et al. ; 2001 <sup>16</sup></a></li><br />
<br />
<li>17 <a href=http://www.ncbi.nlm.nih.gov/pubmed/19784453>Governance of dual-use research: an ethical dilemma. | Selgelid ; 2009 <sup>17</sup></a></li><br />
<br />
<li>18 <a href=http://www.ncbi.nlm.nih.gov/pubmed/18625678>Evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures. | Kimman et al. 2008 <sup>18</sup></a> </li><br />
<br />
<li>19) <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819452>A Hippocratic Oath for life scientists | Revill et Dando ; 2006 <sup>19</sup></a></li><br />
<br />
<li>20 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819443>Empowerment and restraint in scientific communication. New developments make it easier to share information, but more difficult to deal with dual-use biology. | Campbell ; 2006<sup>20</sup></a></li><br />
<br />
<li>21 <a href=http://www.ncbi.nlm.nih.gov/pubmed/16819441>When risk outweighs benefit | Aken ; 2006 <sup>21</sup></a></li><br />
<br />
<li>22 <a href=http://www.ncbi.nlm.nih.gov/pubmed/12789408>Advances in life sciences and bioterrorism. Risks, perspectives and responsibilities.| Beck ; 2003 <sup>22</sup></a></li><br />
<br />
<li>23 <a href=http://www.ncbi.nlm.nih.gov/pubmed/12590130>PNAS policy on publication of sensitive material in the life sciences | Cozzarelli ; 2003 <sup>23</sup></a></li><br />
<br />
<li>24 <a href=http://www.sciencemag.org/cgi/reprint/310/5745/77.pdf>Characterization of the<br />
Reconstructed 1918 Spanish<br />
Influenza Pandemic Virus | Tumpey et al. ; 2005<sup>24</sup></a> </li><br />
<br />
<li>25 <a href=http://www.nature.com/nature/journal/v438/n7065/pdf/438134a.pdf>Deadly flu virus can be<br />
sent through the mail| v. Bubnoff; 2005<sup>25</sup></a></li><br />
<br />
<li>26 <a href=https://static.igem.org/mediawiki/2010/0/09/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_2001.pdf>Risk assessment of human Adeno-associated viruses| ZKBS; 2001<sup>26</sup></a></li><br />
<br />
<li>27 <a href=https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Advises_for_AAV_carrying_cell_cycle_regulating_genes_2004.pdf>Advises for AAV carrying cell cycle regulating genes| ZKBS; 2004 <sup>27</sup></a></li><br />
<br />
<li>28 <a href=https://static.igem.org/mediawiki/2010/d/dd/Freiburg10_Risk_assessment_of_human_Adeno-associated_viruses_and_AAV_derived_vectors_2005.pdf>Risk assessment of human Adeno-associated viruses and AAV derived vectors| ZKBS; 2005 <sup>28</sup></a> </li><br />
<br />
<li>29 <a href=http://www.ncbi.nlm.nih.gov/pubmed/10700178>Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.| Kai et al. ; 2000 <sup>29</sup></a><br />
Bioverteilung in Klinischer Studie </li><br />
<br />
<li>30 <a href=https://static.igem.org/mediawiki/2010/7/76/Freiburg10_Safetyapplication.pdf>Biosafety application of the iGEM team Freiburg_Bioware 2010<sup>30</sup></a> (in German)</li><br />
<br />
<li>31 <a href=https://static.igem.org/mediawiki/2010/1/18/Freiburg10_Safetyconfirmation.jpg>Official classification as Biological Safety Level 1 by the local biosafety office<sup>31</sup></a></li><br />
<br />
<li>32 <a href=http://oba.od.nih.gov/oba/rac/guidelines_02/Appendix_B.htm>Appendix B | National Institute of Health <sup>32</sup></a></li><br />
<br />
</html></div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-10-08T09:06:12Z<p>Kristian: </p>
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<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/87/Virus_homepage.gif" alt="virus" title="virus (3.5MB)" id="animated_virus"/><br />
<p class="standard"><br />
In the last years the number of people affected by cancer increased significantly. About 25% cases of death are caused by malign tumors. Even though the industry is putting great efforts into developing effective strategies against cancer, innovative methods to fight tumors are needed.<br />
This year’s aim of Freiburg Bioware team 2010 is to create a universal virus construction kit which can be designed by submitted BioBricks. Our goal is to knockout the natural tropism of adeno-associated virus particles and to specifically target tumor cells by genetic engineering of the viral surface. The recombinant viruses will contain different genes coding for enzymes which convert harmless drugs into toxic molecules in targeted cell and kill the tumor cells. This approach will be a great advance in improving the growing field of personalized medicine.<br />
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<u2>Virus Construction Kit</u2><br />
<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/b/b2/Freiburg10_Manual-Logo.png" id="ccc" /><br />
<p class="standard"> Text </p><br />
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<u2>Project Summary</u2><br />
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<p class="standard"><br> Klick for more</p><br />
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<p class="standard">Modularisation and Modification of the viral tropismn and the effector function resulted in many different BioBricks.<br> Click here to explore them.</p><br />
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<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/a/ae/Freiburg10_Biosafety.png" id="ccc" /><br />
<p class="standard">The aspect of biological safety was considered well and a risk assessment and security profile for the Adeno-associated Virus were created. To hear about this click here.</p><br />
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<p class="standard">In order to let the iGEM Community grow together we started the Cuckoo Clock Cometition. This Lab-Fun-Photo-Contest intends to give an impression from the work of other Teams. Click to become impressed.</p><br />
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<center><u0>Sponsors</u0></center><br />
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<a href="http://www.roche.com/index.htm" target="_blank"><img src="https://static.igem.org/mediawiki/2010/3/39/Freiburg10_sponsors_Roche.jpg" class="spon5" alt="Roche" width="auto" height="90"/></a><br />
<a href="http://www.qiagen.com/default_qs.aspx" target="_blank"><img src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_sponsors_Qiagen.jpg" class="spon5" alt="Qiagen" width="140px" height="auto"/></a><br />
<a href="http://www.zeiss.de/en" target="_blank"><img src="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_Sponsor_Zeiss.jpg" class="spon5" alt="Zeiss" width="120" height="auto" /></a><br />
<a href="http://www.starlab.de/int/?l=2" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/51/Freiburg10_Sponsor_StarLab.gif" class="spon5" alt="Starlab" width="180px" height="auto"/></a><br /><br />
<a href="http://www.home.agilent.com" target="_blank"><img src="https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_Sponsor_Agilent.jpg" class="spon5" alt="agilent" width="210" height="auto"/></a><br />
<a href="http://www.fermentas.de/index.php?&language=en" target="_blank"><img src="https://static.igem.org/mediawiki/2010/a/aa/Freiburg10_Sponsor_Fermentas.jpg" class="spon5" alt="Fermentas" width="130" height="auto"/></a><br />
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<a href="http://www.peqlab.com" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_Sponsor_Peqlab.gif" width="150px" height="auto" class="spon5" alt="peqlab" /></a><br />
<a href="http://www.geneart.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9a/Freiburg10_Sponsor_Geneart.png" width="140px" height="auto" class="spon5" alt="Geneart" /></a><br />
<a href="http://www.eppendorf.com/int/?l=1&action=start" target="_blank"><img src="https://static.igem.org/mediawiki/2010/d/d2/Freiburg10_Sponsor_eppendorf.jpeg" width="150px" height="auto" class="spon5" alt="Eppendorf" /></a><br />
<a href="http://www.gilson.com/en/" target="_blank"><img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Sponsor_Gilson.jpeg" width="160px" height="auto" class="spon5" alt="Gilson" /></a><br />
<a href="http://www.gatc-biotech.com" target="_blank"><img src="https://static.igem.org/mediawiki/2010/7/74/Freiburg10_Sponsor_GATC.jpg" width="140px" height="auto" class="spon5" alt="GATC" /></a><br />
<a href="https://www.atg-biosynthetics.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_Sponsor_ATG.jpg" width="150px" height="auto" class="spon5" alt="ATG" /></a><br />
<a href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank"><img src="https://static.igem.org/mediawiki/2010/f/f4/Freiburg10_Sponsor_Biozym.png" width="130px" height="auto" class="spon5" alt="Biozym" /></a><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-09-11T19:49:37Z<p>Kristian: </p>
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<a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware"<br />
id="clustrMapsLink"><br />
<img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" <br />
style="border:0px;"<br />
alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg"<br />
onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
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<!-- Main area --><br />
<br />
<div class="box_right"><br />
<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/87/Virus_homepage.gif" alt="virus" title="virus (3.5MB)" id="animated_virus"/><br />
<p class="standard"><br />
In the last years the number of people affected by cancer increased significantly. About 25% cases of death are caused by malign tumors. Even though the industry is putting great efforts into developing effective strategies against cancer, innovative methods to fight tumors are needed.<br />
This year’s aim of Freiburg Bioware team 2010 is to create a universal virus construction kit which can be designed by submitted BioBricks. Our goal is to knockout the natural tropism of adeno-associated virus particles and to specifically target tumor cells by genetic engineering of the viral surface. The recombinant viruses will contain different genes coding for enzymes which convert harmless drugs into toxic molecules in targeted cell and kill the tumor cells. This approach will be a great advance in improving the growing field of personalized medicine.<br />
</p><br />
</div><br />
<div class="box_right"><br />
<div id="boxes_right"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project"><br />
Project summary<br />
</a><br />
</div><br />
<div class="boxes_four"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Team"><br />
Team<br />
</a><br />
</div><br />
<div class="boxes_four"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook"><br />
Notebook<br />
</a><br />
</div><br />
<div class="boxes_four"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/BioBricks"><br />
BioBricks &trade;<br />
</a><br />
</div><br />
<div class="boxes_four"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Safty"><br />
Human Practice<br />
</a><br />
</div><br />
</div><br />
<br />
<!--Sponsors--><br />
<div class="box_right"><br />
<p id="sponsors">Sponsors:</p><br />
</div><br />
<br /><br />
<center><br />
<a href="http://www.uni-freiburg.de/start-en.html?set_language=en" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/uni_logo.jpg" class="spon5" alt="Uni Freiburg" width="auto" height="170"/></a><br />
<a href="http://www.bioss.uni-freiburg.de/cms/index.php#" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/bioss.png" class="spon5" width="auto" height="170" alt="Bioss"/></a><br />
<a href="http://www.molbiotech.uni-freiburg.de/" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/b/b8/Freiburg09_Sponsors.png" class="spon5" width="auto" height="170" alt="kuk.lab"/></a><br />
<br />
<br /><br />
<a href="http://www.roche.com/index.htm" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/roche.png" class="spon5" alt="Roche" width="auto" height="90"/></a><br />
<a href="http://www.daad.de/en/index.html" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/e/e1/Freiburg10_Sponsor_DAAD.jpg" class="spon5" width="auto" height="80" alt="DAAD"/></a><br />
<a href="http://www.starlab.de/int/?l=2" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/5/51/Freiburg10_Sponsor_StarLab.gif" class="spon5" alt="Starlab" width="200px" height="auto"/></a><br />
<a href="http://www.qiagen.com/default_qs.aspx" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/qiagen.png" class="spon5" alt="Qiagen"/></a><br />
<br />
<br /><br />
<a href="http://www.home.agilent.com" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/r1_agilent_logo.gif" class="spon5" alt="agilent" width="210" height="auto"/></a><br />
<a href="http://www.hiss-dx.de/hiss/index.php?id=43&L=1" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/3/32/Hiss.png" class="spon5" alt="Hiss" width="90" height="auto"/></a><br />
<a href="http://www.fermentas.de/index.php?&language=en" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/a/aa/Freiburg10_Sponsor_Fermentas.jpg" class="spon5" alt="Fermentas" width="120" height="auto"/></a><br />
<a href="http://www.zeiss.de/en" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/7/7f/Freiburg10_Sponsor_Zeiss.jpg" class="spon5" alt="Zeiss" width="90" height="auto" /></a><br />
<a href="http://www.geneart.com/" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/9/9a/Freiburg10_Sponsor_Geneart.png" width="140px" height="auto" class="spon5" alt="Geneart" /></a><br />
<a href="http://www.eppendorf.com/int/?l=1&action=start" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/d/d2/Freiburg10_Sponsor_eppendorf.jpeg" width="180px" height="auto" class="spon5" alt="Eppendorf" /></a><br />
<a href="http://www.gilson.com/en/" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/2/2f/Freiburg10_Sponsor_Gilson.gif" width="180px" height="auto" class="spon5" alt="Gilson" /></a><br />
<a href="https://www.atg-biosynthetics.com/" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/f/fb/Freiburg10_Sponsor_ATG.jpg" width="180px" height="auto" class="spon5" alt="ATG" /></a><br />
<a href="http://www.biozym.com/site/21/Produkte.aspx" target="_blank"><img src="http://www.molbiotech.uni-freiburg.de/iGEM/images/Biozym_ScienceIsOurBusiness.jpg" width="130px" height="auto" class="spon5" alt="Biozym" /></a><br />
</center><br />
</html></div>Kristianhttp://2010.igem.org/Team:Freiburg_SoftwareTeam:Freiburg Software2010-08-27T18:31:25Z<p>Kristian: </p>
<hr />
<div><!-- *** What falls between these lines is the Alert Box! You can remove it from your pages once you have read and understood the alert *** --><br />
<br />
<html><br />
<style><br />
body {background:silver;}<br />
p.test {font-size:18px;}<br />
#testa {font-weight:bold;}<br />
a:hover {text-decoration:none;color:red;}<br />
a {color:blue;}<br />
</style><br />
<br /><br />
<center><br />
<p><br />
<br /><br />
<b>Project Description</b><br /><br />
SynBioWave is an open-source, synthetic biological software suite based on Google’s communication tool Wave (wave.google.com). SynBioWave is made for collaborative research comprising parts design and documentation. Moreover, biologists can record and share the process of creating research data and perform basic tasks using SynBioWave. In addition, SynBioWave is a framework for developing Google Wave extensions with a strong focus on synthetic biological functionality. In the Wave, SynBioWave can be used like a gadget.<br />
The last year’s team of Freiburg built the SynBioWave robot and implemented it on the Google Wave server. This year we are going to improve the functions and availability of this innovative software tool. We want to create robots which provide additional and improved functions such as DNA to RNA translation or blast search robots. Furthermore we want to help Google Wave becoming more famous.<br />
Please find more informations at <a href="http://www.synbiowave.org" target="_blank">http://www.synbiowave.org</a>.<br />
</p><br />
<br />
<br />
<br />
<a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/wiki/index.php?title=Team:Freiburg_Software" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/wiki/index.php?title=Team:Freiburg_Software" 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://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a><br />
</center><br />
<br /><br />
<br />
You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2009.igem.org/Help:Template/Examples">HERE</a>.<br />
</div><br />
<br />
<br /><br />
</html><br />
<br />
<!-- *** End of the alert box *** --><br />
<br />
<br />
{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:Freiburg_Software_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Software_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Software | Team Example]]<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Software|Home]]<br />
!align="center"|[[Team:Freiburg_Software/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Software Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Software/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Software/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Software/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Software/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Software/Safety|Safety]]<br />
|}<br />
<br /></div>Kristianhttp://2010.igem.org/Team:Freiburg_SoftwareTeam:Freiburg Software2010-08-27T18:30:45Z<p>Kristian: </p>
<hr />
<div><!-- *** What falls between these lines is the Alert Box! You can remove it from your pages once you have read and understood the alert *** --><br />
<br />
<html><br />
<style><br />
body {background:silver;}<br />
p.test {font-size:18px;}<br />
#testa {font-weight:bold;}<br />
a:hover {text-decoration:none;color:red;}<br />
a {color:blue;}<br />
</style><br />
<br /><br />
<center><br />
<p><br />
<br /><br />
<b>Project Description</b><br /><br />
SynBioWave is an open-source, synthetic biological software suite based on Google’s communication tool Wave (wave.google.com). SynBioWave is made for collaborative research comprising parts design and documentation. Moreover, biologists can record and share the process of creating research data and perform basic tasks using SynBioWave. In addition, SynBioWave is a framework for developing Google Wave extensions with a strong focus on synthetic biological functionality. In the Wave, SynBioWave can be used like a gadget.<br />
The last year’s team of Freiburg built the SynBioWave robot and implemented it on the Google Wave server. This year we are going to improve the functions and availability of this innovative software tool. We want to create robots which provide additional and improved functions such as DNA to RNA translation or blast search robots. Furthermore we want to help Google Wave becoming more famous.<br />
Please find more informations at <a href="http://www.synbiowave.org" target="_blank">http://www.synbiowave.org</a>.<br />
</p><br />
<br />
<br />
<br />
<a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/wiki/index.php?title=Team:Freiburg_Software" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/wiki/index.php?title=Team:Freiburg_Software" 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://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a><br />
</center><br />
<br /><br />
<br />
You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2009.igem.org/Help:Template/Examples">HERE</a>.<br />
</div><br />
<br />
</div><br />
<br /><br />
</html><br />
<br />
<!-- *** End of the alert box *** --><br />
<br />
<br />
{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:Freiburg_Software_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Software_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Software | Team Example]]<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Software|Home]]<br />
!align="center"|[[Team:Freiburg_Software/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Software Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Software/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Software/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Software/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Software/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Software/Safety|Safety]]<br />
|}<br />
<br /></div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-08-27T18:27:21Z<p>Kristian: </p>
<hr />
<div><br><br />
'''Short project description''': <br><br />
In the last years the number of people affected by cancer increased significantly. About 25% cases of death are caused by malign tumors. Even though the industry is putting great efforts into developing effective strategies against cancer, innovative methods to fight tumors are needed. <br><br />
This year’s aim of '''Freiburg Bioware team 2010''' is to create a universal virus construction kit which can be designed by submitted BioBricks. Our goal is to knockout the natural tropism of adeno-associated virus particles and to specifically target tumor cells by genetic engineering of the viral surface. The recombinant viruses will contain different genes coding for enzymes which convert harmless drugs into toxic molecules in targeted cell and kill the tumor cells. This approach will be a great advance in improving the growing field of personalized medicine. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
<a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware"<br />
id="clustrMapsLink"><br />
<img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" <br />
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onerror="this.onerror=null; this.src='http://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
</a><br />
</html><br />
<br />
<br />
<br />
{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:Freiburg_Bioware_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Bioware_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Bioware | Team Example]]<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Bioware|Home]]<br />
!align="center"|[[Team:Freiburg_Bioware/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Bioware Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Bioware/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Bioware/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Bioware/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Bioware/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Bioware/Safety|Safety]]<br />
|}</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-08-27T18:14:00Z<p>Kristian: </p>
<hr />
<div><br><br />
'''Short project description''': <br><br />
In the last years the number of people affected by cancer increased significantly. About 25% cases of death are caused by malign tumors. Even though the industry is putting great efforts into developing effective strategies against cancer, innovative methods to fight tumors are needed. <br><br />
This year’s aim of '''Freiburg Bioware team 2010''' is to create a universal virus construction kit which can be designed by submitted BioBricks. Our goal is to knockout the natural tropism of adeno-associated virus particles and to specifically target tumor cells by genetic engineering of the viral surface. The recombinant viruses will contain different genes coding for enzymes which convert harmless drugs into toxic molecules in targeted cell and kill the tumor cells. This approach will be a great advance in improving the growing field of personalized medicine. <br />
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{|align="justify"<br />
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|[[Image:Freiburg_Bioware_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Bioware_team.png|right|frame|Your team picture]]<br />
|-<br />
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|align="center"|[[Team:Freiburg_Bioware | Team Example]]<br />
|}<br />
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<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Bioware|Home]]<br />
!align="center"|[[Team:Freiburg_Bioware/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Bioware Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Bioware/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Bioware/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Bioware/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Bioware/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Bioware/Safety|Safety]]<br />
|}</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-08-27T18:10:46Z<p>Kristian: </p>
<hr />
<div><br><br />
'''Short project description''': <br><br />
In the last years the number of people affected by cancer increased significantly. About 25% cases of death are caused by malign tumors. Even though the industry is putting great efforts into developing effective strategies against cancer, innovative methods to fight tumors are needed. <br><br />
This year’s aim of '''Freiburg Bioware team 2010''' is to create a universal virus construction kit which can be designed by submitted BioBricks. Our goal is to knockout the natural tropism of adeno-associated virus particles and to specifically target tumor cells by genetic engineering of the viral surface. The recombinant viruses will contain different genes coding for enzymes which convert harmless drugs into toxic molecules in targeted cell and kill the tumor cells. This approach will be a great advance in improving the growing field of personalized medicine. <br />
<br />
<br />
<a href="http://www2.clustrmaps.com/counter/maps.php?url=https://2010.igem.org/Team:Freiburg_Bioware" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/counter/index2.php?url=https://2010.igem.org/Team:Freiburg_Bioware" 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://clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://clustrmaps.com';" /><br />
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{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:Freiburg_Bioware_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Bioware_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Bioware | Team Example]]<br />
|}<br />
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<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Bioware|Home]]<br />
!align="center"|[[Team:Freiburg_Bioware/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Bioware Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Bioware/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Bioware/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Bioware/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Bioware/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Bioware/Safety|Safety]]<br />
|}</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-04-14T20:53:31Z<p>Kristian: </p>
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You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2009.igem.org/Help:Template/Examples">HERE</a>.<br />
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You <strong>MUST</strong> have a team description page, a project abstract, a complete project description, a lab notebook, and a safety page. PLEASE keep all of your pages within your teams namespace. <br />
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{|align="justify"<br />
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|[[Image:Freiburg_Bioware_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Bioware_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Bioware | Team Example]]<br />
|}<br />
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<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Bioware|Home]]<br />
!align="center"|[[Team:Freiburg_Bioware/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Bioware Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Bioware/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Bioware/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Bioware/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Bioware/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Bioware/Safety|Safety]]<br />
|}</div>Kristianhttp://2010.igem.org/IGEM_workshop_proposal_pageIGEM workshop proposal page2010-04-01T20:27:14Z<p>Kristian: </p>
<hr />
<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year, we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de university] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus, we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]; <html><script src="http://www.gmodules.com/ig/ifr?url=http://hosting.gmodules.com/ig/gadgets/file/114281111391296844949/driving-directions.xml&amp;up_fromLocation=&amp;up_myLocations=Freiburg%20im%20Breisgau%2C%20Germany&amp;synd=open&amp;w=320&amp;h=55&amp;title=Here+you+can+get+driving+directions&amp;brand=light&amp;lang=en&amp;country=US&amp;border=%23ffffff%7C3px%2C1px+solid+%23999999&amp;output=js"></script></script></html><br />
<br />
We propose to use the hotel [http://www.hotel-stadt-freiburg.de/index.php?id=343&L=1 "Hotel Stadt Freiburg"], which is conveniently located next to the medical campus of the university.<br><br />
[[Image:Map_Hotel-Stadt-Freiburg.png|200px]]<br />
<br><br />
If you have questions or comments regarding the Freiburg proposal, please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/File:Map_Hotel-Stadt-Freiburg.pngFile:Map Hotel-Stadt-Freiburg.png2010-04-01T20:18:23Z<p>Kristian: uploaded a new version of "Image:Map Hotel-Stadt-Freiburg.png"</p>
<hr />
<div></div>Kristianhttp://2010.igem.org/File:Map_Hotel-Stadt-Freiburg.pngFile:Map Hotel-Stadt-Freiburg.png2010-04-01T20:12:16Z<p>Kristian: </p>
<hr />
<div></div>Kristianhttp://2010.igem.org/IGEM_workshop_proposal_pageIGEM workshop proposal page2010-04-01T20:10:38Z<p>Kristian: /* Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010 */</p>
<hr />
<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year, we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de university] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus, we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]; <html><script src="http://www.gmodules.com/ig/ifr?url=http://hosting.gmodules.com/ig/gadgets/file/114281111391296844949/driving-directions.xml&amp;up_fromLocation=&amp;up_myLocations=Freiburg%20im%20Breisgau%2C%20Germany&amp;synd=open&amp;w=320&amp;h=55&amp;title=Here+you+can+get+driving+directions&amp;brand=light&amp;lang=en&amp;country=US&amp;border=%23ffffff%7C3px%2C1px+solid+%23999999&amp;output=js"></script></script></html><br />
<br />
We propose to use the hotel [http://www.hotel-stadt-freiburg.de/index.php?id=343&L=1 "Hotel Stadt Freiburg"], which is conveniently located next to the medical campus of the university.<br />
[[Image:Map_Hotel-Stadt-Freiburg|200px]]<br />
<br />
If you have questions or comments regarding the Freiburg proposal, please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/IGEM_workshop_proposal_pageIGEM workshop proposal page2010-04-01T19:56:25Z<p>Kristian: New page: ==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010== Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospi...</p>
<hr />
<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year, we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de university] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus, we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]; <html><script src="http://www.gmodules.com/ig/ifr?url=http://hosting.gmodules.com/ig/gadgets/file/114281111391296844949/driving-directions.xml&amp;up_fromLocation=&amp;up_myLocations=Freiburg%20im%20Breisgau%2C%20Germany&amp;synd=open&amp;w=320&amp;h=55&amp;title=Here+you+can+get+driving+directions&amp;brand=light&amp;lang=en&amp;country=US&amp;border=%23ffffff%7C3px%2C1px+solid+%23999999&amp;output=js"></script></script></html><br />
<br />
If you have questions or comments regarding the Freiburg proposal, please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/Team:Freiburg_BiowareTeam:Freiburg Bioware2010-04-01T19:55:25Z<p>Kristian: </p>
<hr />
<div><br />
<br><br />
<b>[[iGEM workshop proposal page]]</b><br><br />
<br />
<br />
<!-- *** What falls between these lines is the Alert Box! You can remove it from your pages once you have read and understood the alert *** --><br />
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</div><br />
<div id="instructions" style="text-align: center; font-weight: normal; font-size: small; color: #f6f6f6; padding: 5px;"><br />
You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2009.igem.org/Help:Template/Examples">HERE</a>.<br />
</div><br />
<div id="warning" style="text-align: center; font-weight: bold; font-size: small; color: #f6f6f6; padding: 5px;"><br />
You <strong>MUST</strong> have a team description page, a project abstract, a complete project description, a lab notebook, and a safety page. PLEASE keep all of your pages within your teams namespace. <br />
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</html><br />
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<!-- *** End of the alert box *** --><br />
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<br />
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<br />
{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:Freiburg_Bioware_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:Freiburg_Bioware_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:Freiburg_Bioware | Team Example]]<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Freiburg_Bioware|Home]]<br />
!align="center"|[[Team:Freiburg_Bioware/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Freiburg_Bioware Official Team Profile]<br />
!align="center"|[[Team:Freiburg_Bioware/Project|Project]]<br />
!align="center"|[[Team:Freiburg_Bioware/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Freiburg_Bioware/Modeling|Modeling]]<br />
!align="center"|[[Team:Freiburg_Bioware/Notebook|Notebook]]<br />
!align="center"|[[Team:Freiburg_Bioware/Safety|Safety]]<br />
|}</div>Kristianhttp://2010.igem.org/Workshops/ProposalWorkshops/Proposal2010-01-25T13:57:13Z<p>Kristian: /* Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010 */</p>
<hr />
<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year, we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de university] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus, we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]; <html><script src="http://www.gmodules.com/ig/ifr?url=http://hosting.gmodules.com/ig/gadgets/file/114281111391296844949/driving-directions.xml&amp;up_fromLocation=&amp;up_myLocations=Freiburg%20im%20Breisgau%2C%20Germany&amp;synd=open&amp;w=320&amp;h=55&amp;title=Here+you+can+get+driving+directions&amp;brand=light&amp;lang=en&amp;country=US&amp;border=%23ffffff%7C3px%2C1px+solid+%23999999&amp;output=js"></script></script></html><br />
<br />
If you have questions or comments regarding the Freiburg proposal, please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/Workshops/ProposalWorkshops/Proposal2010-01-25T12:33:03Z<p>Kristian: /* Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010 */</p>
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<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year, we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de university] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus, we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]<br />
<br />
If you have questions or comments regarding the Freiburg proposal, please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/Workshops/ProposalWorkshops/Proposal2010-01-25T11:05:12Z<p>Kristian: New page: ==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010== Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospi...</p>
<hr />
<div>==Proposal: Freiburg, Germany, as a host for the European iGEM Spring Workshop 2010==<br />
<br />
Freiburg participated in the iGEM competition three times and very much enjoyed the quality and hospitality of the previous workshops in Zurich, Paris, and London. This year we would like to give back to the community and host the spring workshop. As a [http://www.uni-freiburg.de University] with over 20 000 students, we are well equipped with various sizes of lecture halls. The [http://www.bioss.uni-freiburg.de/cms/index.php "Centre for Biological Signalling Studies" (bioss)] will sponsor the event, and thus we can provide subsidized accommodation.<br />
<br />
Information about Freiburg can be found on [http://en.wikipedia.org/wiki/Freiburg_im_Breisgau wikipedia] the [http://www.freiburg.de/servlet/PB/menu/1140679_l2/index.html official city web-site] or [http://wikitravel.org/en/Freiburg wikitravel].<br />
<br />
Freiburg is strategically located in the middle of Europe with high speed train connection (e.g. 3:40 min to Paris) and several airports close by. It is only a 50 min bus drive from the Euro airport (Basel-Mulhouse-Freiburg airport), which hosts major airlines and the discount airline EasyJet. The Karlsruhe/Baden-Baden airport (Baden airpark) can be reached by a 1 h bus or train ride and is served by the discount airlines Ryanair and Airberlin. Frankfurt international airport is connected by a non-stop 2 h train ride. Also the airports of Stuttgart and Zurich can be reached in approximately 2 h and Strasbourg in about 1 h.<br />
<br />
[http://www.easyjet.com Easyjet] serving Euroairport: [[Image:easyjet_euroairport.jpg|200px]]; [http://www.airberlin.com Airberlin] serving Badenairpark: [[Image:Airberlin_badenairpark_.jpg|200px]];<br />
<br />
[http://www.ryanair.com Ryanair] serving Badenairpark: [[Image:ryanair_badenairpark.jpg|200px]]<br />
<br />
If you have questions or comments regarding the Freiburg proposal please contact: [mailto:kristian@biologie.uni-freiburg.de?Subject=iGEM_workshop2010 Kristian Müller]<br />
----</div>Kristianhttp://2010.igem.org/File:Easyjet_euroairport.jpgFile:Easyjet euroairport.jpg2010-01-25T10:07:15Z<p>Kristian: </p>
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<div></div>Kristianhttp://2010.igem.org/File:Ryanair_badenairpark.jpgFile:Ryanair badenairpark.jpg2010-01-25T09:58:38Z<p>Kristian: </p>
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<div></div>Kristianhttp://2010.igem.org/File:Airberlin_badenairpark_.jpgFile:Airberlin badenairpark .jpg2010-01-25T09:55:13Z<p>Kristian: </p>
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<div></div>Kristianhttp://2010.igem.org/File:Easyjet_Basel.jpgFile:Easyjet Basel.jpg2010-01-25T09:43:01Z<p>Kristian: </p>
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<div></div>Kristian