http://2013.igem.org/wiki/index.php?title=Special:Contributions/FlorianA&feed=atom&limit=50&target=FlorianA&year=&month=2013.igem.org - User contributions [en]2024-03-29T08:52:58ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T19:31:47Z<p>FlorianA: /* Achievements */</p>
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<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
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== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
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<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
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== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><br />
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<a href="/File:TUM13_blubbern-stop.gif" class="image"><br />
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</a><br />
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<a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><br />
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /><br />
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Hover over the picture!<br />Then you can actually <b>HEAR</b> the sound in our laboratory.<br />
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<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project Competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
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== Sponsors ==<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T19:29:34Z<p>FlorianA: /* Achievements */</p>
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<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><br />
<div class="thumbinner" style="width:442px;"><br />
<a href="/File:TUM13_blubbern-stop.gif" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="400" class="thumbimage" /><br />
</a><br />
<div class="thumbcaption"><br />
<div class="magnify"><br />
<a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><br />
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /><br />
</a><br />
</div><br />
Hover over the picture!<br />Then you can actually <b>HEAR</b> the sound in our laboratory.<br />
</div><br />
</div><br />
</div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project Competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
<html><br />
<a class="sponsor" href="http://www.tum.de/"><img src="https://static.igem.org/mediawiki/2013/9/97/Logo_TU-Muenchen_01.png" width="190px"></a><br />
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<a class="sponsor" href="http://www.daad.de/de/index.html"><img src="https://static.igem.org/mediawiki/2013/f/f7/TUM13_DAAD.jpg" width="120px"></a><br />
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<a class="sponsor" href="http://www.promega.de/"><img src="https://static.igem.org/mediawiki/2013/f/f0/Promega-300.jpg" width="175px"></a><br />
<a class="sponsor" href="http://www.qiagen.com/"><img src="https://static.igem.org/mediawiki/2013/6/60/TUM13_Sponsor_Qiagen.png" width="100px"></a> <br />
<a class="sponsor" href="http://www.iba-lifesciences.com/"><img src="https://static.igem.org/mediawiki/2013/9/9c/Iba_logo_claim_r_farbe.jpg" width="180px"></a> <br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/TUM13_ScriptTeam:TU-Munich/TUM13 Script2014-05-09T19:27:55Z<p>FlorianA: </p>
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"https://static.igem.org/mediawiki/2013/5/5f/TUM13_tour-start-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/6/6e/TUM13_tour-previous-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/3/35/TUM13_tour-next-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/4/44/TUM13_button-application-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/a/a7/TUM13_button-autoannotator-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/d/d5/TUM13_button-physco-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/e/e6/TUM13_button-entrepreneur-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/0/01/TUM13_button-safety-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/d/d6/TUM13_button-judging-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/c/c5/TUM13_gotop-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/2/2d/TUM13_gotop.png",<br />
"https://static.igem.org/mediawiki/2013/b/b4/TUM13_ajax-loading.gif",<br />
"https://static.igem.org/mediawiki/2013/2/29/TUM13_ajax-bg.png" );<br />
<br />
// preload footer links<br />
<br />
preload( "https://static.igem.org/mediawiki/2013/0/05/TUM13_address-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/c/c7/TUM13_contact-icon-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/9/9f/TUM13_facebook-icon-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/2/22/TUM13_twitter-icon-highlight.png",<br />
"https://static.igem.org/mediawiki/2013/a/a1/TUM13_youtube-icon-highlight.png" );<br />
<br />
// preload team pictures<br />
<br />
if ( $("div#teamfield").length > 0 ) {<br />
preload( "https://static.igem.org/mediawiki/2013/9/95/TUM13_moos.png",<br />
"https://static.igem.org/mediawiki/2013/b/b6/TUM13_katrin-front.png", // Katrin<br />
"https://static.igem.org/mediawiki/2013/1/13/TUM13_katrin-t.png",<br />
"https://static.igem.org/mediawiki/2013/a/a8/TUM13_katrin-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/7/72/TUM13_katrin-l.png",<br />
"https://static.igem.org/mediawiki/2013/7/7f/TUM13_katrin-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/c/cf/TUM13_katrin-b.png",<br />
"https://static.igem.org/mediawiki/2013/2/28/TUM13_katrin-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/3/3e/TUM13_katrin-r.png",<br />
"https://static.igem.org/mediawiki/2013/8/8e/TUM13_katrin-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/b/b6/TUM13_rosario-front.png", // Rosario<br />
"https://static.igem.org/mediawiki/2013/2/2e/TUM13_rosario-t.png",<br />
"https://static.igem.org/mediawiki/2013/4/42/TUM13_rosario-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/9/91/TUM13_rosario-l.png",<br />
"https://static.igem.org/mediawiki/2013/6/66/TUM13_rosario-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/0/05/TUM13_rosario-b.png",<br />
"https://static.igem.org/mediawiki/2013/8/8e/TUM13_rosario-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/1/1a/TUM13_rosario-r.png",<br />
"https://static.igem.org/mediawiki/2013/3/38/TUM13_rosario-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/5/55/TUM13_fabian-front.png", // Fabian<br />
"https://static.igem.org/mediawiki/2013/0/0a/TUM13_fabian-t.png",<br />
"https://static.igem.org/mediawiki/2013/8/87/TUM13_fabian-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/b/b7/TUM13_fabian-l.png",<br />
"https://static.igem.org/mediawiki/2013/0/0b/TUM13_fabian-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/2/24/TUM13_fabian-b.png",<br />
"https://static.igem.org/mediawiki/2013/5/51/TUM13_fabian-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/0/01/TUM13_fabian-r.png",<br />
"https://static.igem.org/mediawiki/2013/5/51/TUM13_fabian-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/d/de/TUM13_andreas-front.png", // Andreas<br />
"https://static.igem.org/mediawiki/2013/6/67/TUM13_andreas-t.png",<br />
"https://static.igem.org/mediawiki/2013/d/da/TUM13_andreas-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/8/84/TUM13_andreas-l.png",<br />
"https://static.igem.org/mediawiki/2013/d/d6/TUM13_andreas-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/9/9c/TUM13_andreas-b.png",<br />
"https://static.igem.org/mediawiki/2013/2/21/TUM13_andreas-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/5/55/TUM13_andreas-r.png",<br />
"https://static.igem.org/mediawiki/2013/8/81/TUM13_andreas-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/5/50/TUM13_louise-front.png", // Louise<br />
"https://static.igem.org/mediawiki/2013/5/58/TUM13_louise-t.png",<br />
"https://static.igem.org/mediawiki/2013/8/86/TUM13_louise-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/4/4f/TUM13_louise-l.png",<br />
"https://static.igem.org/mediawiki/2013/b/b1/TUM13_louise-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/a/ad/TUM13_louise-b.png",<br />
"https://static.igem.org/mediawiki/2013/3/3f/TUM13_louise-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/b/bc/TUM13_louise-r.png",<br />
"https://static.igem.org/mediawiki/2013/f/fe/TUM13_louise-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/4/47/TUM13_johanna-front.png", // Johanna<br />
"https://static.igem.org/mediawiki/2013/f/fc/TUM13_johanna-t.png",<br />
"https://static.igem.org/mediawiki/2013/1/10/TUM13_johanna-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/8/8c/TUM13_johanna-l.png",<br />
"https://static.igem.org/mediawiki/2013/b/b1/TUM13_johanna-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/4/4a/TUM13_johanna-b.png",<br />
"https://static.igem.org/mediawiki/2013/3/32/TUM13_johanna-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/8/89/TUM13_johanna-r.png",<br />
"https://static.igem.org/mediawiki/2013/d/d2/TUM13_johanna-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/2/29/TUM13_meike-front.png", // Meike<br />
"https://static.igem.org/mediawiki/2013/8/85/TUM13_meike-t.png",<br />
"https://static.igem.org/mediawiki/2013/3/3e/TUM13_meike-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/0/07/TUM13_meike-l.png",<br />
"https://static.igem.org/mediawiki/2013/7/79/TUM13_meike-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/6/63/TUM13_meike-b.png",<br />
"https://static.igem.org/mediawiki/2013/a/af/TUM13_meike-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/e/e3/TUM13_meike-r.png",<br />
"https://static.igem.org/mediawiki/2013/c/c1/TUM13_meike-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/d/db/TUM13_volker-front.png", // Volker<br />
"https://static.igem.org/mediawiki/2013/1/1a/TUM13_volker-t.png",<br />
"https://static.igem.org/mediawiki/2013/0/0a/TUM13_volker-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/4/4f/TUM13_volker-l.png",<br />
"https://static.igem.org/mediawiki/2013/f/fb/TUM13_volker-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/5/57/TUM13_volker-b.png",<br />
"https://static.igem.org/mediawiki/2013/8/86/TUM13_volker-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/2/2c/TUM13_volker-r.png",<br />
"https://static.igem.org/mediawiki/2013/2/27/TUM13_volker-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/3/3c/TUM13_polte-front.png", // Polte<br />
"https://static.igem.org/mediawiki/2013/9/9e/TUM13_polte-t.png",<br />
"https://static.igem.org/mediawiki/2013/9/9c/TUM13_polte-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/0/03/TUM13_polte-l.png",<br />
"https://static.igem.org/mediawiki/2013/5/58/TUM13_polte-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/9/99/TUM13_polte-b.png",<br />
"https://static.igem.org/mediawiki/2013/0/0f/TUM13_polte-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/b/b9/TUM13_polte-r.png",<br />
"https://static.igem.org/mediawiki/2013/6/61/TUM13_polte-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/d/da/TUM13_leonie-front.png", // Leonie<br />
"https://static.igem.org/mediawiki/2013/7/79/TUM13_leonie-t.png",<br />
"https://static.igem.org/mediawiki/2013/9/9c/TUM13_leonie-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/7/78/TUM13_leonie-l.png",<br />
"https://static.igem.org/mediawiki/2013/2/26/TUM13_leonie-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/8/8a/TUM13_leonie-b.png",<br />
"https://static.igem.org/mediawiki/2013/c/cc/TUM13_leonie-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/8/81/TUM13_leonie-r.png",<br />
"https://static.igem.org/mediawiki/2013/2/25/TUM13_leonie-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/0/0c/TUM13_philipp-front.png", // Philipp<br />
"https://static.igem.org/mediawiki/2013/a/a4/TUM13_philipp-t.png",<br />
"https://static.igem.org/mediawiki/2013/2/2d/TUM13_philipp-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/c/cf/TUM13_philipp-l.png",<br />
"https://static.igem.org/mediawiki/2013/8/82/TUM13_philipp-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/3/3a/TUM13_philipp-b.png",<br />
"https://static.igem.org/mediawiki/2013/1/1e/TUM13_philipp-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/f/ff/TUM13_philipp-r.png",<br />
"https://static.igem.org/mediawiki/2013/6/68/TUM13_philipp-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/2/25/TUM13_jeff-front.png", // Jeff<br />
"https://static.igem.org/mediawiki/2013/4/41/TUM13_jeff-t.png",<br />
"https://static.igem.org/mediawiki/2013/9/90/TUM13_jeff-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/2/21/TUM13_jeff-l.png",<br />
"https://static.igem.org/mediawiki/2013/b/bf/TUM13_jeff-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/e/ec/TUM13_jeff-b.png",<br />
"https://static.igem.org/mediawiki/2013/f/f4/TUM13_jeff-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/3/3e/TUM13_jeff-r.png",<br />
"https://static.igem.org/mediawiki/2013/a/a5/TUM13_jeff-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/a/a1/TUM13_chris-front.png", // Chris<br />
"https://static.igem.org/mediawiki/2013/4/42/TUM13_chris-t.png",<br />
"https://static.igem.org/mediawiki/2013/a/a4/TUM13_chris-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/4/4d/TUM13_chris-l.png",<br />
"https://static.igem.org/mediawiki/2013/e/ec/TUM13_chris-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/9/9b/TUM13_chris-b.png",<br />
"https://static.igem.org/mediawiki/2013/c/c6/TUM13_chris-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/7/72/TUM13_chris-r.png",<br />
"https://static.igem.org/mediawiki/2013/0/05/TUM13_chris-t-r.png",<br />
"https://static.igem.org/mediawiki/2013/0/03/TUM13_flo-front.png", // Flo<br />
"https://static.igem.org/mediawiki/2013/b/b4/TUM13_flo-t.png",<br />
"https://static.igem.org/mediawiki/2013/c/ce/TUM13_flo-t-l.png",<br />
"https://static.igem.org/mediawiki/2013/0/06/TUM13_flo-l.png",<br />
"https://static.igem.org/mediawiki/2013/1/11/TUM13_flo-b-l.png",<br />
"https://static.igem.org/mediawiki/2013/6/6b/TUM13_flo-b.png",<br />
"https://static.igem.org/mediawiki/2013/4/4b/TUM13_flo-b-r.png",<br />
"https://static.igem.org/mediawiki/2013/3/30/TUM13_flo-r.png"/*,<br />
"https://static.igem.org/mediawiki/2013/5/5d/TUM13_jeff-anim.gif", // Gifs<br />
"https://static.igem.org/mediawiki/2013/f/f9/TUM13_rosario-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/d/d1/TUM13_fabian-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/a/aa/TUM13_philipp-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/f/ff/TUM13_johanna-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/a/a5/TUM13_andi-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/e/ea/TUM13_flo-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/6/6c/TUM13_polte-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/7/70/TUM13_leonie-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/2/24/TUM13_louise-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/9/9b/TUM13_chris-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/6/6c/TUM13_katrin-anim.gif",<br />
"https://static.igem.org/mediawiki/2013/5/5e/TUM13_meike-anim.gif" */ );<br />
}<br />
<br />
// Slideshows<br />
<br />
$('.bxslider').bxSlider({<br />
responsive: false,<br />
auto: true,<br />
autoHover: true,<br />
captions: true<br />
});<br />
<br />
$('.bxgallery').bxSlider({<br />
captions: true,<br />
slideMargin: 10,<br />
minSlides: 3,<br />
maxSlides: 3,<br />
moveSlides: 1,<br />
slideWidth: 5000<br />
});<br />
<br />
// Lightbox<br />
<br />
$("div.thumbinner > a img").slimbox({}, function(el) {<br />
url = el.src;<br />
if (url.indexOf('thumb') != -1) {<br />
url = url.substring(0, url.lastIndexOf('/'));<br />
url = url.replace('/thumb/', '/');<br />
}<br />
description = $(el).parents("div.thumbinner").children("div.thumbcaption").text();<br />
return [url, description];<br />
}, function(el) {<br />
return (this == el);<br />
});<br />
<br />
$("ul.bxgallery img").slimbox({ loop: true }, function(el) {<br />
url = el.src;<br />
if (url.indexOf('thumb') != -1) {<br />
url = url.substring(0, url.lastIndexOf('/'));<br />
url = url.replace('/thumb/', '/');<br />
}<br />
description = el.title + el.alt;<br />
return [url, description];<br />
}, function(el) {<br />
return (this == el) || (this.parentNode.parentNode && (this.parentNode.parentNode == el.parentNode.parentNode));<br />
});<br />
<br />
<br />
// Counter and Countdown<br />
<br />
function render_counter(c) {<br />
i = 0;<br />
iid = window.setInterval(function(){<br />
if ( (c-i) > (c/200) ) {<br />
$('span#counter').html(i);<br />
i += Math.round(c/200);<br />
} else {<br />
$('span#counter').html(c);<br />
window.clearInterval(iid);<br />
}<br />
}, 10);<br />
}<br />
<br />
if ($('span#counter').length > 0) {<br />
$.ajax({<br />
url: "https://2013.igem.org/Special:PopularPages",<br />
success: function( html ) {<br />
dom = $.parseHTML(html);<br />
visitors = $(dom).find('a[title="Team:TU-Munich"]').parent().text();<br />
visitors = visitors.substring(visitors.indexOf('(')+1);<br />
visitors = visitors.substring(0, visitors.indexOf(' '));<br />
visitors = visitors.replace(',', '');<br />
render_counter(visitors);<br />
},<br />
error: function( xhr, status ) {<br />
render_counter(4700);<br />
}<br />
});<br />
}<br />
<br />
if ($('span#countdown').length > 0) {<br />
clock = window.setInterval(function(){<br />
jetzt = new Date();<br />
time_left = Date.UTC(2013, 9, 29, 4, 0, 0) - Date.UTC(jetzt.getUTCFullYear(), jetzt.getUTCMonth(), jetzt.getUTCDate(), jetzt.getUTCHours(), jetzt.getUTCMinutes(), jetzt.getUTCSeconds());<br />
time_left = (time_left < 0) ? 0 : time_left ;<br />
left_sec = (time_left/1000)%60;<br />
left_sec = (left_sec < 10) ? "0" + left_sec : left_sec;<br />
left_min = Math.floor(time_left/60000)%60;<br />
left_min = (left_min < 10) ? "0" + left_min : left_min;<br />
left_h = Math.floor(time_left/3600000)%24;<br />
left_h = (left_h < 10) ? "0" + left_h : left_h;<br />
left_d = Math.floor(time_left/86400000);<br />
left_d = (left_d == 1) ? left_d + " day" : left_d + " days";<br />
$('span#countdown').html(left_d + " " + left_h + ":" + left_min + ":" + left_sec);<br />
}, 1000);<br />
}<br />
<br />
<br />
// Animate teamfield<br />
<br />
if ( $("div#teamfield").length > 0 ) {<br />
<br />
var $members = $("div#teamfield a");<br />
<br />
$("body").mousemove(function(event){<br />
for (i=0; i<$members.length; i++) {<br />
<br />
if ( $members.eq(i).offset().left > event.pageX ) {<br />
<br />
if ( $members.eq(i).offset().top > event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("top-left");<br />
<br />
} else if ( $members.eq(i).offset().top <= event.pageY && ( $members.eq(i).offset().top + $members.eq(i).height() ) >= event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("left");<br />
<br />
} else if ( ( $members.eq(i).offset().top + $members.eq(i).height() ) < event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("bottom-left");<br />
<br />
}<br />
<br />
} else if ( $members.eq(i).offset().left <= event.pageX && ( $members.eq(i).offset().left + $members.eq(i).width() ) >= event.pageX ) {<br />
<br />
if ( $members.eq(i).offset().top > event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("top");<br />
<br />
} else if ( $members.eq(i).offset().top <= event.pageY && ( $members.eq(i).offset().top + $members.eq(i).height() ) >= event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("front");<br />
<br />
} else if ( ( $members.eq(i).offset().top + $members.eq(i).height() ) < event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("bottom");<br />
<br />
} <br />
<br />
} else if ( ( $members.eq(i).offset().left + $members.eq(i).width() ) < event.pageX ) {<br />
<br />
if ( $members.eq(i).offset().top > event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("top-right");<br />
<br />
} else if ( $members.eq(i).offset().top <= event.pageY && ( $members.eq(i).offset().top + $members.eq(i).height() ) >= event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("right");<br />
<br />
} else if ( ( $members.eq(i).offset().top + $members.eq(i).height() ) < event.pageY ) {<br />
<br />
$members.eq(i).removeClass();<br />
$members.eq(i).addClass("bottom-right");<br />
<br />
} <br />
<br />
}<br />
<br />
}<br />
});<br />
<br />
// team gifs<br />
/*<br />
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}<br />
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*/<br />
// name generator<br />
<br />
function swaffel() {<br />
var volker = [ 'Molker Votorrad', 'Swolker', 'Vollgas Motorrad', 'Schmolkers Vorrat', 'Polter Volker', 'Volkan Murrat', 'Voll der Koller', 'Mullah Murrat', 'Mordsding Vollhart', 'Volter Streichzart', 'Vollkorn Mossart', 'Vortex Mozart', 'Vodka Melonath', 'Volker Racho', 'Molkers Vorrath', 'Herbert' , 'Volker Willesso'];<br />
index = Math.round(Math.random() * volker.length);<br />
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<br />
$(document).ready(myWikiReady);</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T19:25:53Z<p>FlorianA: /* Achievements */</p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
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<div id="wikicontent-container"><br />
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<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
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<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
<a class="button safety" href="https://2013.igem.org/Team:TU-Munich/Project/Safety">Safety</a><br />
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<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><br />
<div class="thumbinner" style="width:442px;"><br />
<a href="/File:TUM13_blubbern-stop.gif" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="442" class="thumbimage" /><br />
</a><br />
<div class="thumbcaption"><br />
<div class="magnify"><br />
<a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><br />
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /><br />
</a><br />
</div><br />
Hover over the picture!<br />Then you can actually <b>HEAR</b> the sound in our laboratory.<br />
</div><br />
</div><br />
</div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project Competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
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<a class="sponsor" href="http://eu.idtdna.com/site/"><img src="https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://www.thermoscientific.com"><img src="https://static.igem.org/mediawiki/2013/e/ed/TUM13_Sponsor_logo_Thermo.jpg" width="140px"></a><br />
<a class="sponsor" href="http://www.erasynbio.eu/"><img src="https://static.igem.org/mediawiki/2013/5/58/Logo_ERASynBio.png" width="180px"></a><br />
<div class="visualClear"></div><br />
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<!-- Ende des Inhalts --><br />
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<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T19:22:49Z<p>FlorianA: </p>
<hr />
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{{Team:TU-Munich/TUM13_Style}}<br />
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<span id="countdown" style="color: #502204; font: normal 24px/40px sketch_rockwell,Arial,sans-serif;"></span> left to World Wiki Freeze<br />
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<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
</div><br />
<div style="height: 145px; margin: 5px 0px 5px;"><br />
<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
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</div><br />
</html><br />
<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><br />
<div class="thumbinner" style="width:442px;"><br />
<a href="/File:TUM13_blubbern-stop.gif" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="442" class="thumbimage" /><br />
</a><br />
<div class="thumbcaption"><br />
<div class="magnify"><br />
<a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><br />
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /><br />
</a><br />
</div><br />
Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer<br />
</div><br />
</div><br />
</div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project Competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
<html><br />
<a class="sponsor" href="http://www.tum.de/"><img src="https://static.igem.org/mediawiki/2013/9/97/Logo_TU-Muenchen_01.png" width="190px"></a><br />
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<a class="sponsor" href="http://www.daad.de/de/index.html"><img src="https://static.igem.org/mediawiki/2013/f/f7/TUM13_DAAD.jpg" width="120px"></a><br />
<a class="sponsor" href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="190px"></a><br />
<a class="sponsor" href="http://www.eurofins.de/"><img src="https://static.igem.org/mediawiki/2013/b/bc/TUM_Eurofins.png" width="200px"></a><br />
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<a class="sponsor" href="http://www.neb.com/"><img src="https://static.igem.org/mediawiki/2013/5/50/TUM13_Sponsor_NEB.jpg" width="155px"></a><br />
<a class="sponsor" href="http://www.promega.de/"><img src="https://static.igem.org/mediawiki/2013/f/f0/Promega-300.jpg" width="175px"></a><br />
<a class="sponsor" href="http://www.qiagen.com/"><img src="https://static.igem.org/mediawiki/2013/6/60/TUM13_Sponsor_Qiagen.png" width="100px"></a> <br />
<a class="sponsor" href="http://www.iba-lifesciences.com/"><img src="https://static.igem.org/mediawiki/2013/9/9c/Iba_logo_claim_r_farbe.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://eu.idtdna.com/site/"><img src="https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://www.thermoscientific.com"><img src="https://static.igem.org/mediawiki/2013/e/ed/TUM13_Sponsor_logo_Thermo.jpg" width="140px"></a><br />
<a class="sponsor" href="http://www.erasynbio.eu/"><img src="https://static.igem.org/mediawiki/2013/5/58/Logo_ERASynBio.png" width="180px"></a><br />
<div class="visualClear"></div><br />
<a class="sponsor" style="float: none; margin: 10px auto;" href="http://www.roche.com/"><img src="https://static.igem.org/mediawiki/2013/d/d2/TUM13_Roche.jpg" height="80px"></a><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T19:21:16Z<p>FlorianA: /* Achievements */</p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
<br />
<div id="wikicontent-container"><br />
<div id="wikicontent"><html><ul class="bxslider"> <!-- das muss so hässlich sein, damit kein absatz eingefügt wird --><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/97/TUM13_slider_team1.jpg" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/3/30/TUM13_slider_team2.jpg" /></li><br />
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<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
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<div style="height: 145px; margin: 5px 0px 5px;"><br />
<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
<a class="button safety" href="https://2013.igem.org/Team:TU-Munich/Project/Safety">Safety</a><br />
<a class="button judging" href="https://2013.igem.org/Team:TU-Munich/Team/Judging">Judging</a><br />
</div><br />
</html><br />
<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><br />
<div class="thumbinner" style="width:442px;"><br />
<a href="/File:TUM13_blubbern-stop.gif" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="450" class="thumbimage" /><br />
</a><br />
<div class="thumbcaption"><br />
<div class="magnify"><br />
<a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><br />
<img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /><br />
</a><br />
</div><br />
Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer<br />
</div><br />
</div><br />
</div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
<html><br />
<a class="sponsor" href="http://www.tum.de/"><img src="https://static.igem.org/mediawiki/2013/9/97/Logo_TU-Muenchen_01.png" width="190px"></a><br />
<a class="sponsor" href="http://wzw.tum.de/"><img src="https://static.igem.org/mediawiki/2013/a/ac/Wzwlogo2.png" width="210px"></a><br />
<a class="sponsor" href="http://biologische-chemie.userweb.mwn.de/index.html"><img src="https://static.igem.org/mediawiki/2013/0/05/Unbenannt.PNG" width="190px"></a><br />
<a class="sponsor" href="http://www.daad.de/de/index.html"><img src="https://static.igem.org/mediawiki/2013/f/f7/TUM13_DAAD.jpg" width="120px"></a><br />
<a class="sponsor" href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="190px"></a><br />
<a class="sponsor" href="http://www.eurofins.de/"><img src="https://static.igem.org/mediawiki/2013/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a class="sponsor" href="http://www.mathworks.de/"><img src="https://static.igem.org/mediawiki/2013/0/0c/TUM13_MathWorks.png" width="200px"></a><br />
<a class="sponsor" href="http://www.ika.com/"><img src="https://static.igem.org/mediawiki/2013/9/9d/TUM13_IKA.png" width="105px"></a><br />
<a class="sponsor" href="http://www.neb.com/"><img src="https://static.igem.org/mediawiki/2013/5/50/TUM13_Sponsor_NEB.jpg" width="155px"></a><br />
<a class="sponsor" href="http://www.promega.de/"><img src="https://static.igem.org/mediawiki/2013/f/f0/Promega-300.jpg" width="175px"></a><br />
<a class="sponsor" href="http://www.qiagen.com/"><img src="https://static.igem.org/mediawiki/2013/6/60/TUM13_Sponsor_Qiagen.png" width="100px"></a> <br />
<a class="sponsor" href="http://www.iba-lifesciences.com/"><img src="https://static.igem.org/mediawiki/2013/9/9c/Iba_logo_claim_r_farbe.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://eu.idtdna.com/site/"><img src="https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://www.thermoscientific.com"><img src="https://static.igem.org/mediawiki/2013/e/ed/TUM13_Sponsor_logo_Thermo.jpg" width="140px"></a><br />
<a class="sponsor" href="http://www.erasynbio.eu/"><img src="https://static.igem.org/mediawiki/2013/5/58/Logo_ERASynBio.png" width="180px"></a><br />
<div class="visualClear"></div><br />
<a class="sponsor" style="float: none; margin: 10px auto;" href="http://www.roche.com/"><img src="https://static.igem.org/mediawiki/2013/d/d2/TUM13_Roche.jpg" height="80px"></a><br />
<br />
<center style="margin-top: 10px;"><a href="http://www2.clustrmaps.com/user/c81109e0d" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/stats/maps-no_clusters/2013.igem.org-Team-TU-Munich-thumb.jpg" 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';" width="150" /></a></center><br />
</html><br />
<br />
<!-- Ende des Inhalts --><br />
</div><br />
</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/TUM13_wiki.cssTeam:TU-Munich/TUM13 wiki.css2014-05-09T19:11:38Z<p>FlorianA: </p>
<hr />
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<br />
/* Fix AutoAnnotator */<br />
div#wikicontent table#AutoAnnotator, div#wikicontent table#AutoAnnotator table {<br />
background: #ffffff;<br />
border: 1px solid #000000;<br />
font-size: 12px;<br />
line-height: 18px;<br />
margin-bottom: 0px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator td, div#wikicontent table#AutoAnnotator th {<br />
border: 1px solid #000000;<br />
padding: 1px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder th {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder th {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator th {<br />
background: rgb(221, 221, 221);<br />
}<br />
<br />
div#wikicontent div#autoannotator-computing {<br />
text-align: center;<br />
}<br />
<br />
/* Fix parts table */<br />
div#groupparts {<br />
width: 920px !important;<br />
}<br />
<br />
div#groupparts td {<br />
background: #ded6ca;<br />
}<br />
<br />
/* Sponsors */<br />
div#wikicontent a.sponsor {<br />
display: block;<br />
float: left;<br />
width: 286px;<br />
height: 100px;<br />
margin: 10px;<br />
background: #FFFFFF;<br />
position: relative;<br />
}<br />
<br />
div#wikicontent a.sponsor img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
bottom: 0;<br />
margin: auto;<br />
}<br />
<br />
div#wikicontent div.sponsor {<br />
background: #ded6ca;<br />
height: 215px;<br />
margin-bottom: 20px;<br />
padding: 0px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.sponsor a.sponsor {<br />
width: 320px;<br />
height: 215px;<br />
margin: 0 1em 0 0;<br />
}<br />
<br />
div#wikicontent div.sponsor p {<br />
padding: 1em;<br />
}<br />
<br />
div div#wikicontent div.overview {<br />
height: 240px;<br />
width: 440px;<br />
padding: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview img {<br />
float: right;<br />
margin: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview h3 {<br />
margin: 5px 0px 0px;<br />
clear: none;<br />
}<br />
<br />
div#wikicontent div.overview span.editsection {<br />
display: none;<br />
}<br />
<br />
/* Boxes */<br />
div#wikicontent div.box-right {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 0px 20px 10px;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
}<br />
<br />
div#wikicontent div.box-left {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 10px 20px 0px;<br />
padding: 10px 10px 0px;<br />
clear: left;<br />
float: left;<br />
}<br />
<br />
div#wikicontent div.team.box-left, div#wikicontent div.team.box-right {<br />
height: 620px;<br />
}<br />
<br />
div#wikicontent div.team div, div#wikicontent div.team a img {<br />
float: right;<br />
position: relative;<br />
margin: 0 0 10px 10px;<br />
background: url('https://static.igem.org/mediawiki/2013/4/4d/TUM13_computing.gif') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div.team div.portrait {<br />
width: 200px;<br />
height: 356px;<br />
}<br />
<br />
div#wikicontent div.team div.landscape {<br />
width: 300px;<br />
height: 169px;<br />
}<br />
<br />
div#wikicontent div.team div img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
}<br />
<br />
div#wikicontent div.team div img.top {<br />
z-index: 20;<br />
}<br />
<br />
div#wikicontent div.team div:hover img.top {<br />
visibility: hidden;<br />
}<br />
<br />
div#wikicontent div.team div img.bottom {<br />
z-index: 10;<br />
}<br />
<br />
div#wikicontent div.box-definition {<br />
width: 320px;<br />
background: #ded6ca;<br />
margin: 0 0 1em 1em;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent div.box-definition p {<br />
margin-bottom: 10px;<br />
}<br />
<br />
div#wikicontent div.box-center {<br />
background: #ded6ca;<br />
margin-bottom: 20px;<br />
padding: 10px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.team h4 {<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent div.quote {<br />
margin: 1em 1.5em;<br />
padding: 0.5em 0.7em;<br />
background: #ded6ca;<br />
}<br />
<br />
div#wikicontent div.quote p {<br />
margin: 0px;<br />
font: normal 20px/30px Garamond, Baskerville, "Baskerville Old Face", "Hoefler Text", "Times New Roman", serif;<br />
/* font-style: italic; */<br />
}<br />
<br />
div#wikicontent div.quote div.author {<br />
text-align: right;<br />
font-variant: small-caps;<br />
}<br />
<br />
/* Buttons */<br />
<br />
div#wikicontent a.tour {<br />
display: block;<br />
font: normal 24px/40px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.tour.start {<br />
float: right;<br />
width: 205px;<br />
height: 40px;<br />
padding: 5px 7px 5px 38px;<br />
background: url('https://static.igem.org/mediawiki/2013/3/36/TUM13_tour-start.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.start:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/5/5f/TUM13_tour-start-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous {<br />
float: left;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 5px 5px 25px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/f/fc/TUM13_tour-previous.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/6/6e/TUM13_tour-previous-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next {<br />
float: right;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 25px 5px 5px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/c/c6/TUM13_tour-next.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/3/35/TUM13_tour-next-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button {<br />
display: block;<br />
float: left;<br />
width: 131px;<br />
height: 20px;<br />
padding: 11px 7px 114px;<br />
margin-left: 10px;<br />
font: normal 16px/20px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.button.application {<br />
margin-left: 0px;<br />
background: url('https://static.igem.org/mediawiki/2013/7/7b/TUM13_button-application.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.application:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/4/44/TUM13_button-application-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator {<br />
background: url('https://static.igem.org/mediawiki/2013/4/43/TUM13_button-autoannotator.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a7/TUM13_button-autoannotator-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco {<br />
background: url('https://static.igem.org/mediawiki/2013/7/77/TUM13_button-physco.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d5/TUM13_button-physco-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e1/TUM13_button-entrepreneur.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e6/TUM13_button-entrepreneur-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4f/TUM13_button-safety.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/01/TUM13_button-safety-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging {<br />
background: url('https://static.igem.org/mediawiki/2013/4/47/TUM13_button-judging.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d6/TUM13_button-judging-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay {<br />
width: 100%;<br />
height: 100%;<br />
z-index: 9996;<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
background: url('https://static.igem.org/mediawiki/2013/2/29/TUM13_ajax-bg.png') repeat 0% 0%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay img {<br />
position: absolute;<br />
z-index: 9997;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
margin: auto;<br />
}<br />
<br />
/* End content style ==================================================== */<br />
<br />
<br />
/* Start footer style =================================================== */<br />
<br />
/* Makes the footer stick to the bottom */<br />
html, body {<br />
height: 100%;<br />
} <br />
<br />
body > div#globalWrapper {<br />
position: relative;<br />
height: auto;<br />
min-height: 100%;<br />
margin: 0px auto -156px;<br />
}<br />
<br />
div#globalWrapper > div.visualClear {<br />
height: 156px;<br />
}<br />
<br />
div#footer-box {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c3/TUM13_footer-bg.png') repeat-x 50% 0%;<br />
padding: 5px 0px 10px;<br />
height: 141px;<br />
width: 100%;<br />
position: absolute;<br />
bottom: 0px;<br />
color: #d4c6b6;<br />
font-weight: normal;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#social-footer {<br />
width: 940px;<br />
height: 95px;<br />
margin: 0px auto;<br />
padding: 0px 10px 10px;<br />
}<br />
<br />
div#social-footer div#annotator, div#social-footer div#address, div#social-footer div#follow {<br />
width: 293px;<br />
height: 95px;<br />
margin: 0px 10px;<br />
float: left;<br />
}<br />
<br />
div#social-footer h2 {<br />
font: normal 28px/42px sketch_rockwell, Arial, sans-serif;<br />
text-align: center;<br />
margin: 0px;<br />
}<br />
<br />
div#social-footer div#annotator {<br />
text-align: center;<br />
}<br />
<br />
div#social-footer div#annotator input {<br />
font-size: 12px;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#social-footer div#annotator input[type="text"] {<br />
margin: 0px 0px 5px;<br />
border: 1px solid #502204;<br />
background: #d4c6b6;<br />
width: 275px;<br />
}<br />
<br />
div#social-footer div#address p {<br />
margin: 0px;<br />
width: 50%;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow a {<br />
display: block;<br />
width: 32px;<br />
height: 32px;<br />
margin: 9px;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow div {<br />
margin: 3px 21px 0px;<br />
}<br />
<br />
div#social-footer div#follow a#adress {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4b/TUM13_address-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#adress:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/05/TUM13_address-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_contact-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c7/TUM13_contact-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook {<br />
background: url('https://static.igem.org/mediawiki/2013/c/cb/TUM13_facebook-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/9/9f/TUM13_facebook-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter {<br />
background: url('https://static.igem.org/mediawiki/2013/3/34/TUM13_twitter-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/2/22/TUM13_twitter-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube {<br />
background: url('https://static.igem.org/mediawiki/2013/4/49/TUM13_youtube-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a1/TUM13_youtube-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#footer {<br />
width: 920px;<br />
margin: 0px auto;<br />
padding: 0px 20px;<br />
text-align: center;<br />
}<br />
<br />
div#footer ul {<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
div#footer li {<br />
padding: 0px 5px;<br />
display: inline;<br />
}<br />
<br />
div#footer li#t-recentchangeslinked {<br />
display: none;<br />
}<br />
<br />
div#footer-box a {<br />
color: inherit;<br />
}<br />
<br />
div#footer-box a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
div#footer div#f-poweredbyico {<br />
margin-top: 3px;<br />
float: right;<br />
}<br />
<br />
div#footer div#f-copyrightico {<br />
margin-top: 3px;<br />
float: left;<br />
}<br />
<br />
/* End footer style ===================================================== */</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/TUM13_wiki.cssTeam:TU-Munich/TUM13 wiki.css2014-05-09T19:07:05Z<p>FlorianA: </p>
<hr />
<div>/* iGEM TU-Munich Stylesheet by Florian Albrecht (albrecht.flo@gmx.net)<br />
if you want to use parts of this on your wiki, cite us and / or write an e-mail to us */<br />
<br />
/* Start general style ================================================== */<br />
<br />
/* Add Sketch-rockwell for menu and headings (hosted on dropbox.com) */<br />
@font-face {<br />
font-family: 'sketch_rockwell';<br />
src: url('https://dl.dropboxusercontent.com/s/c2su1zy22xs6x9k/sketchrockwell-bold-webfont.eot');<br />
src: url('https://dl.dropboxusercontent.com/s/c2su1zy22xs6x9k/sketchrockwell-bold-webfont.eot?#iefix') format('embedded-opentype'),<br />
url('https://dl.dropboxusercontent.com/s/gnktv67746ccx42/sketchrockwell-bold-webfont.woff') format('woff'),<br />
url('https://dl.dropboxusercontent.com/s/b6g9lp8shab463r/sketchrockwell-bold-webfont.ttf') format('truetype'),<br />
url('https://dl.dropboxusercontent.com/s/cbfupthn2al3xk1/sketchrockwell-bold-webfont.svg#sketch_rockwellregular') format('svg');<br />
font-weight: normal;<br />
font-style: normal;<br />
}<br />
<br />
@media screen and (-webkit-min-device-pixel-ratio:0) {<br />
@font-face {<br />
font-family: 'sketch_rockwell';<br />
src: url('https://dl.dropboxusercontent.com/s/cbfupthn2al3xk1/sketchrockwell-bold-webfont.svg#sketch_rockwellregular') format('svg');<br />
}<br />
}<br />
<br />
/* Beige noisy bg and default font */<br />
html body {<br />
background: #ded6ca url('https://static.igem.org/mediawiki/2013/a/a3/TUM13_paper-bg.png') repeat;<br />
color: #000000;<br />
font-family: Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
/* Upper brown and green bg */<br />
div#globalWrapper {<br />
background: url('https://static.igem.org/mediawiki/2013/0/0c/TUM13_green-bg.png') repeat-x 50% 0%;<br />
}<br />
<br />
/* End general style ==================================================== */<br />
<br />
<br />
/* Start top-section style ============================================== */<br />
<br />
/* Remove logo and search */<br />
div#p-logo, div#search-controls {<br />
display: none;<br />
}<br />
<br />
/* Center wiki-menu; set font & bg (as fallback) color */<br />
div#top-section {<br />
margin: 0px auto;<br />
width: 930px;<br />
height: 20px;<br />
color: #d4c6b6;<br />
background: #411c03 url('https://static.igem.org/mediawiki/2013/b/b3/TUM13_brown-top-bg.png') repeat-x 50% 0%;<br />
}<br />
<br />
/* Set font and positions */<br />
div#menubar {<br />
font-weight: bold;<br />
font-size: 13px;<br />
line-height: 20px;<br />
}<br />
<br />
div#menubar.right-menu {<br />
float: right;<br />
}<br />
<br />
div#menubar.left-menu {<br />
float: left;<br />
}<br />
<br />
/* Fix text */<br />
div#menubar.left-menu a, div#menubar.left-menu li {<br />
text-transform: capitalize;<br />
}<br />
<br />
div#menubar li {<br />
padding: 0px 5px;<br />
display: inline;<br />
}<br />
<br />
/* White on hover */<br />
div#menubar a:hover {<br />
color: #FFFFFF;<br />
text-decoration: none;<br />
}<br />
<br />
div#menubar li[style="color:white;cursor:default"] {<br />
display: none;<br />
}<br />
<br />
/* End top-section style ================================================ */<br />
<br />
<br />
/* Start header style =================================================== */<br />
<br />
/* Hide page title (it would appear before the header) */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
/* Header-bg and positioning */<br />
div#header-container {<br />
background: url('https://static.igem.org/mediawiki/2013/b/bf/TUM13_page-bg-top.png') no-repeat 50% 0%;<br />
height: 217px;<br />
padding-top: 30px;<br />
}<br />
<br />
/* Header positioning */<br />
div#header {<br />
width: 920px;<br />
height: 130px;<br />
margin: 0px auto;<br />
padding: 5px 20px 0px;<br />
overflow: visible;<br />
}<br />
<br />
/* Logos */<br />
div#header img#physco-logo {<br />
display: block;<br />
height: 130px;<br />
margin-left: 303px;<br />
}<br />
<br />
div#header img#igem-logo {<br />
display: block;<br />
margin-top: 40px;<br />
height: 95px;<br />
float: right;<br />
}<br />
<br />
div#header img#tum-logo {<br />
display: block;<br />
margin-top: 15px;<br />
height: 120px;<br />
float: left;<br />
}<br />
<br />
/* End header style ===================================================== */<br />
<br />
<br />
/* Start menu style ===================================================== */<br />
<br />
/* Menu-bg and spacing*/<br />
div#menu {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a8/TUM13_menu-bg.png') no-repeat 50% 0%;<br />
height: 60px;<br />
padding: 5px 0px 17px;<br />
}<br />
<br />
div#menu ul {<br />
list-style: none;<br />
padding: 0;<br />
margin: 0;<br />
}<br />
<br />
/* Position menu and set font color */<br />
div#menu > ul {<br />
margin: 0px auto;<br />
width: 960px;<br />
height: 60px;<br />
color: #411c03;<br />
}<br />
<br />
/* Position menu elements and set font */<br />
div#menu > ul > li {<br />
float: left;<br />
width: 137px;<br />
height: 44px;<br />
padding: 8px 0px 8px;<br />
text-align: center;<br />
position: relative;<br />
font: normal 24px/44px sketch_rockwell,Arial,sans-serif;<br />
}<br />
<br />
div#menu > ul > li > a {<br />
display: block;<br />
width: 137px;<br />
height: 44px;<br />
color: inherit;<br />
}<br />
<br />
/* Hover effect */<br />
div#menu > ul > li > a:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/9/95/TUM13_menu-highlight.png') repeat-x 50% 50%;<br />
}<br />
<br />
div#menu > ul > li > a:hover {<br />
text-decoration: none;<br />
}<br />
<br />
/* Style and hide submenu */<br />
div#menu > ul > li > ul {<br />
border-left: solid 1px #411c03;<br />
border-right: solid 1px #411c03;<br />
width: 135px;<br />
background: #7cac2c;<br />
display: none;<br />
position: absolute;<br />
top: 60px;<br />
z-index: 9998;<br />
}<br />
<br />
/* Style submenu elements */<br />
div#menu > ul > li > ul > li {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4e/TUM13_submenu-bg.png') repeat-y 50% 50%;<br />
padding: 0px 10px;<br />
text-align: left;<br />
border-bottom: solid 1px #411c03;<br />
font: bold 13px/20px Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
div#menu > ul > li > ul > li > a {<br />
display: block;<br />
width: 115px;<br />
padding: 5px 0px;<br />
color: inherit;<br />
}<br />
<br />
/* Hover effect */<br />
div#menu > ul > li > ul > li:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/db/TUM13_submenu-highlight.png') repeat-y 50% 50%;<br />
}<br />
<br />
div#menu > ul > li > ul > li > a:hover {<br />
text-decoration: none;<br />
}<br />
<br />
/* Show submenu on hover */<br />
div#menu > ul > li:hover > ul {<br />
display: block;<br />
}<br />
<br />
/* End menu style ======================================================= */<br />
<br />
<br />
/* Start content style ================================================== */<br />
<br />
div#bodyContent > p, div#contentSub, div#catlinks, table#toc.toc {<br />
display: none;<br />
}<br />
/*<br />
div#wikicontent > table#toc.toc {<br />
width: 350px;<br />
position: fixed;<br />
top: 100px;<br />
left: -300px;<br />
z-index: 10001;<br />
}<br />
<br />
<br />
div#wikicontent > table#toc.toc:hover {<br />
left: 0px;<br />
}<br />
*/<br />
/* top button */<br />
div#social-footer a#gotop {<br />
display: block;<br />
width: 40px;<br />
height: 40px;<br />
padding: 10px;<br />
background: url('https://static.igem.org/mediawiki/2013/2/2d/TUM13_gotop.png') no-repeat 50% 50%;<br />
position: fixed;<br />
bottom: 15px;<br />
right: 15px;<br />
}<br />
<br />
div#social-footer a#gotop:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c5/TUM13_gotop-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
/* Set page-bg */<br />
div#wikicontent-container {<br />
background: url('https://static.igem.org/mediawiki/2013/a/ac/TUM13_page-bg-middle.png') repeat-y 50% 0%;<br />
padding: 2px 0px 1px;<br />
}<br />
<br />
div#wikicontent > :first-child {<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent > :last-child {<br />
margin-bottom: 0px;<br />
}<br />
<br />
/* Font and position */<br />
div#wikicontent {<br />
font-size: 14px;<br />
line-height: 21px;<br />
background: #e9e3dd;<br />
width: 920px;<br />
margin: 0px auto;<br />
padding: 0px 20px;<br />
text-align: justify;<br />
position: relative;<br />
}<br />
<br />
/* Lower page border */<br />
div#bodyContent > div.visualClear {<br />
background: url('https://static.igem.org/mediawiki/2013/6/6e/TUM13_page-bg-bottom.png') no-repeat 50% 0%;<br />
height: 50px;<br />
}<br />
<br />
/* Standard elements */<br />
div#wikicontent p {<br />
margin: 0 0 1em;<br />
}<br />
<br />
div#wikicontent ul {<br />
padding-left: 2em;<br />
list-style: disc outside;<br />
}<br />
<br />
div#wikicontent div.achievements ul {<br />
list-style: none;<br />
padding-left: 0.4em;<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent div.achievements ul li {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_checkmark-small.png') no-repeat 0% 50%;<br />
padding-left: 30px;<br />
margin: 0px;<br />
line-height: 32px;<br />
}<br />
<br />
div#wikicontent div.achievements h3, div#wikicontent div.achievements h4, div#wikicontent div.achievements h5, div#wikicontent div.achievements h6 {<br />
clear: none;<br />
}<br />
<br />
div#wikicontent ol {<br />
padding-left: 2em;<br />
list-style: decimal outside;<br />
}<br />
<br />
div#wikicontent li {<br />
margin-bottom: 0.5em;<br />
}<br />
<br />
div#wikicontent a {<br />
color: #7cac2c;<br />
}<br />
<br />
div#bodyContent a[href^="https://"], div#bodyContent a[href^="mailto:"], #bodyContent .link-https {<br />
background: transparent;<br />
padding: 0px;<br />
}<br />
<br />
div#wikicontent a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
/* Headings */<br />
div#wikicontent h1, div#wikicontent h2, div#wikicontent h3, div#wikicontent h4, div#wikicontent h5, div#wikicontent h6 {<br />
font-weight: bold;<br />
line-height: 1.5;<br />
margin: 1em 0em 0.5em;<br />
clear: both;<br />
text-align: left;<br />
}<br />
<br />
div#wikicontent .editsection {<br />
color: #7cac2c;<br />
font-family: Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
div#wikicontent h1, div#wikicontent h2 {<br />
color: #502204;<br />
font: normal 2em/1.35 sketch_rockwell,Arial,sans-serif;<br />
border-bottom: 2px solid #502204;<br />
}<br />
<br />
div#wikicontent h3 {<br />
font-size: 1.5em;<br />
}<br />
<br />
div#wikicontent h4 {<br />
font-size: 1.25em;<br />
}<br />
<br />
div#wikicontent h5, div#wikicontent h6 {<br />
font-size: 1.0em;<br />
}<br />
<br />
/* Pictures */<br />
div#wikicontent div.thumb {<br />
border: none;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner {<br />
background: #ded6ca;<br />
border: none;<br />
font-size: 12px;<br />
line-height: 23px;<br />
overflow: hidden;<br />
padding: 5px 4px 2px !important;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner a img {<br />
border: none;<br />
padding-bottom: 3px;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption {<br />
background: transparent;<br />
font-size: inherit;<br />
line-height: inherit;<br />
overflow: hidden;<br />
padding: 0px 5px !important;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption div.magnify {<br />
display: none;<br />
}<br />
<br />
div#wikicontent div.tright {<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent div.tleft {<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
div#wikicontent div.center div.tnone {<br />
clear: both;<br />
margin: 0 0 1em;<br />
}<br />
<br />
/* Tables */<br />
div#wikicontent table {<br />
background: #ded6ca;<br />
border: 1px solid #502204;<br />
margin: 0 auto 1em;<br />
}<br />
<br />
div#wikicontent table.left {<br />
float: left;<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
<br />
div#wikicontent table.right {<br />
float: right;<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent table.safety td, div#wikicontent table.safety th {<br />
text-align: left;<br />
vertical-align: top;<br />
}<br />
<br />
div#wikicontent table caption {<br />
text-align: left;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent td, div#wikicontent th {<br />
border: 1px solid #502204;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#wikicontent th {<br />
background: #d4c6b6;<br />
font-weight: bold;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent table code {<br />
background: transparent;<br />
}<br />
<br />
/* Fix AutoAnnotator */<br />
div#wikicontent table#AutoAnnotator, div#wikicontent table#AutoAnnotator table {<br />
background: #ffffff;<br />
border: 1px solid #000000;<br />
font-size: 12px;<br />
line-height: 18px;<br />
margin-bottom: 0px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator td, div#wikicontent table#AutoAnnotator th {<br />
border: 1px solid #000000;<br />
padding: 1px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder th {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder th {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator th {<br />
background: rgb(221, 221, 221);<br />
}<br />
<br />
div#wikicontent div#autoannotator-computing {<br />
text-align: center;<br />
}<br />
<br />
/* Fix parts table */<br />
div#groupparts {<br />
width: 920px !important;<br />
}<br />
<br />
div#groupparts td {<br />
background: #ded6ca;<br />
}<br />
<br />
/* Sponsors */<br />
div#wikicontent a.sponsor {<br />
display: block;<br />
float: left;<br />
width: 286px;<br />
height: 100px;<br />
margin: 10px;<br />
background: #FFFFFF;<br />
position: relative;<br />
}<br />
<br />
div#wikicontent a.sponsor img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
bottom: 0;<br />
margin: auto;<br />
}<br />
<br />
div#wikicontent div.sponsor {<br />
background: #ded6ca;<br />
height: 215px;<br />
margin-bottom: 20px;<br />
padding: 0px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.sponsor a.sponsor {<br />
width: 320px;<br />
height: 215px;<br />
margin: 0 1em 0 0;<br />
}<br />
<br />
div#wikicontent div.sponsor p {<br />
padding: 1em;<br />
}<br />
<br />
div div#wikicontent div.overview {<br />
height: 240px;<br />
width: 440px;<br />
padding: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview img {<br />
float: right;<br />
margin: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview h3 {<br />
margin: 5px 0px 0px;<br />
clear: none;<br />
}<br />
<br />
div#wikicontent div.overview span.editsection {<br />
display: none;<br />
}<br />
<br />
/* Boxes */<br />
div#wikicontent div.box-right {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 0px 20px 10px;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
}<br />
<br />
div#wikicontent div.box-left {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 10px 20px 0px;<br />
padding: 10px 10px 0px;<br />
clear: left;<br />
float: left;<br />
}<br />
<br />
div#wikicontent div.team.box-left, div#wikicontent div.team.box-right {<br />
height: 620px;<br />
}<br />
<br />
div#wikicontent div.team div, div#wikicontent div.team a img {<br />
float: right;<br />
position: relative;<br />
margin: 0 0 10px 10px;<br />
background: url('https://static.igem.org/mediawiki/2013/4/4d/TUM13_computing.gif') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div.team div.portrait {<br />
width: 200px;<br />
height: 356px;<br />
}<br />
<br />
div#wikicontent div.team div.landscape {<br />
width: 300px;<br />
height: 169px;<br />
}<br />
<br />
div#wikicontent div.team div img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
}<br />
<br />
div#wikicontent div.team div img.top {<br />
z-index: 20;<br />
}<br />
<br />
div#wikicontent div.team div:hover img.top {<br />
visibility: hidden;<br />
}<br />
<br />
div#wikicontent div.team div img.bottom {<br />
z-index: 10;<br />
}<br />
<br />
div#wikicontent div.box-definition {<br />
width: 320px;<br />
background: #ded6ca;<br />
margin: 0 0 1em 1em;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent div.box-definition p {<br />
margin-bottom: 10px;<br />
}<br />
<br />
div#wikicontent div.box-center {<br />
background: #ded6ca;<br />
margin-bottom: 20px;<br />
padding: 10px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.team h4 {<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent div.quote {<br />
margin: 1em 1.5em;<br />
padding: 0.5em 0.7em;<br />
background: #ded6ca;<br />
}<br />
<br />
div#wikicontent div.quote p {<br />
margin: 0px;<br />
font: normal 20px/30px Garamond, Baskerville, "Baskerville Old Face", "Hoefler Text", "Times New Roman", serif;<br />
/* font-style: italic; */<br />
}<br />
<br />
div#wikicontent div.quote div.author {<br />
text-align: right;<br />
font-variant: small-caps;<br />
}<br />
<br />
/* Buttons */<br />
<br />
div#wikicontent a.tour {<br />
display: block;<br />
font: normal 24px/40px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.tour.start {<br />
float: right;<br />
width: 205px;<br />
height: 40px;<br />
padding: 5px 7px 5px 38px;<br />
background: url('https://static.igem.org/mediawiki/2013/3/36/TUM13_tour-start.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.start:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/5/5f/TUM13_tour-start-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous {<br />
float: left;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 5px 5px 25px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/f/fc/TUM13_tour-previous.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/6/6e/TUM13_tour-previous-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next {<br />
float: right;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 25px 5px 5px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/c/c6/TUM13_tour-next.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/3/35/TUM13_tour-next-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button {<br />
display: block;<br />
float: left;<br />
width: 131px;<br />
height: 20px;<br />
padding: 11px 7px 114px;<br />
margin-left: 10px;<br />
font: normal 16px/20px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.button.application {<br />
margin-left: 0px;<br />
background: url('https://static.igem.org/mediawiki/2013/7/7b/TUM13_button-application.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.application:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/4/44/TUM13_button-application-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator {<br />
background: url('https://static.igem.org/mediawiki/2013/4/43/TUM13_button-autoannotator.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a7/TUM13_button-autoannotator-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco {<br />
background: url('https://static.igem.org/mediawiki/2013/7/77/TUM13_button-physco.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d5/TUM13_button-physco-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e1/TUM13_button-entrepreneur.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e6/TUM13_button-entrepreneur-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4f/TUM13_button-safety.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/01/TUM13_button-safety-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging {<br />
background: url('https://static.igem.org/mediawiki/2013/4/47/TUM13_button-judging.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d6/TUM13_button-judging-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay {<br />
width: 100%;<br />
height: 100%;<br />
z-index: 9996;<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
background: url('https://static.igem.org/mediawiki/2013/2/29/TUM13_ajax-bg.png') repeat 0% 0%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay img {<br />
position: absolute;<br />
z-index: 9997;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
margin: auto;<br />
}<br />
<br />
/* End content style ==================================================== */<br />
<br />
<br />
/* Start footer style =================================================== */<br />
<br />
/* Makes the footer stick to the bottom */<br />
html, body {<br />
height: 100%;<br />
} <br />
<br />
body > div#globalWrapper {<br />
position: relative;<br />
height: auto;<br />
min-height: 100%;<br />
margin: 0px auto -156px;<br />
}<br />
<br />
div#globalWrapper > div.visualClear {<br />
height: 156px;<br />
}<br />
<br />
div#footer-box {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c3/TUM13_footer-bg.png') repeat-x 50% 0%;<br />
padding: 5px 0px 10px;<br />
height: 141px;<br />
width: 100%;<br />
position: absolute;<br />
bottom: 0px;<br />
color: #d4c6b6;<br />
font-weight: normal;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#social-footer {<br />
width: 940px;<br />
height: 95px;<br />
margin: 0px auto;<br />
padding: 0px 10px 10px;<br />
}<br />
<br />
div#social-footer div#annotator, div#social-footer div#address, div#social-footer div#follow {<br />
width: 293px;<br />
height: 95px;<br />
margin: 0px 10px;<br />
float: left;<br />
}<br />
<br />
div#social-footer h2 {<br />
font: normal 28px/42px sketch_rockwell, Arial, sans-serif;<br />
text-align: center;<br />
margin: 0px;<br />
}<br />
<br />
div#social-footer div#annotator {<br />
text-align: center;<br />
}<br />
<br />
div#social-footer div#annotator input {<br />
font-size: 12px;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#social-footer div#annotator input[type="text"] {<br />
margin: 0px 0px 5px;<br />
border: 1px solid #502204;<br />
background: #d4c6b6;<br />
width: 275px;<br />
}<br />
<br />
div#social-footer div#address p {<br />
margin: 0px;<br />
width: 50%;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow a {<br />
display: block;<br />
width: 32px;<br />
height: 32px;<br />
margin: 9px;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow div {<br />
margin: 3px 21px 0px;<br />
}<br />
<br />
div#social-footer div#follow a#adress {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4b/TUM13_address-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#adress:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/05/TUM13_address-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_contact-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c7/TUM13_contact-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook {<br />
background: url('https://static.igem.org/mediawiki/2013/c/cb/TUM13_facebook-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/9/9f/TUM13_facebook-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter {<br />
background: url('https://static.igem.org/mediawiki/2013/3/34/TUM13_twitter-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/2/22/TUM13_twitter-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube {<br />
background: url('https://static.igem.org/mediawiki/2013/4/49/TUM13_youtube-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a1/TUM13_youtube-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#footer {<br />
width: 920px;<br />
margin: 0px auto;<br />
padding: 0px 20px;<br />
text-align: center;<br />
}<br />
<br />
div#footer ul {<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
div#footer li {<br />
padding: 0px 5px;<br />
display: inline;<br />
}<br />
<br />
div#footer li#t-recentchangeslinked {<br />
display: none;<br />
}<br />
<br />
div#footer-box a {<br />
color: inherit;<br />
}<br />
<br />
div#footer-box a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
div#footer div#f-poweredbyico {<br />
margin-top: 3px;<br />
float: right;<br />
}<br />
<br />
div#footer div#f-copyrightico {<br />
margin-top: 3px;<br />
float: left;<br />
}<br />
<br />
/* End footer style ===================================================== */</div>FlorianAhttp://2013.igem.org/File:TUM13_checkmark-small.pngFile:TUM13 checkmark-small.png2014-05-09T18:55:28Z<p>FlorianA: uploaded a new version of &quot;File:TUM13 checkmark-small.png&quot;</p>
<hr />
<div></div>FlorianAhttp://2013.igem.org/Team:TU-Munich/TUM13_wiki.cssTeam:TU-Munich/TUM13 wiki.css2014-05-09T18:47:22Z<p>FlorianA: </p>
<hr />
<div>/* iGEM TU-Munich Stylesheet by Florian Albrecht (albrecht.flo@gmx.net)<br />
if you want to use parts of this on your wiki, cite us and / or write an e-mail to us */<br />
<br />
/* Start general style ================================================== */<br />
<br />
/* Add Sketch-rockwell for menu and headings (hosted on dropbox.com) */<br />
@font-face {<br />
font-family: 'sketch_rockwell';<br />
src: url('https://dl.dropboxusercontent.com/s/c2su1zy22xs6x9k/sketchrockwell-bold-webfont.eot');<br />
src: url('https://dl.dropboxusercontent.com/s/c2su1zy22xs6x9k/sketchrockwell-bold-webfont.eot?#iefix') format('embedded-opentype'),<br />
url('https://dl.dropboxusercontent.com/s/gnktv67746ccx42/sketchrockwell-bold-webfont.woff') format('woff'),<br />
url('https://dl.dropboxusercontent.com/s/b6g9lp8shab463r/sketchrockwell-bold-webfont.ttf') format('truetype'),<br />
url('https://dl.dropboxusercontent.com/s/cbfupthn2al3xk1/sketchrockwell-bold-webfont.svg#sketch_rockwellregular') format('svg');<br />
font-weight: normal;<br />
font-style: normal;<br />
}<br />
<br />
@media screen and (-webkit-min-device-pixel-ratio:0) {<br />
@font-face {<br />
font-family: 'sketch_rockwell';<br />
src: url('https://dl.dropboxusercontent.com/s/cbfupthn2al3xk1/sketchrockwell-bold-webfont.svg#sketch_rockwellregular') format('svg');<br />
}<br />
}<br />
<br />
/* Beige noisy bg and default font */<br />
html body {<br />
background: #ded6ca url('https://static.igem.org/mediawiki/2013/a/a3/TUM13_paper-bg.png') repeat;<br />
color: #000000;<br />
font-family: Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
/* Upper brown and green bg */<br />
div#globalWrapper {<br />
background: url('https://static.igem.org/mediawiki/2013/0/0c/TUM13_green-bg.png') repeat-x 50% 0%;<br />
}<br />
<br />
/* End general style ==================================================== */<br />
<br />
<br />
/* Start top-section style ============================================== */<br />
<br />
/* Remove logo and search */<br />
div#p-logo, div#search-controls {<br />
display: none;<br />
}<br />
<br />
/* Center wiki-menu; set font & bg (as fallback) color */<br />
div#top-section {<br />
margin: 0px auto;<br />
width: 930px;<br />
height: 20px;<br />
color: #d4c6b6;<br />
background: #411c03 url('https://static.igem.org/mediawiki/2013/b/b3/TUM13_brown-top-bg.png') repeat-x 50% 0%;<br />
}<br />
<br />
/* Set font and positions */<br />
div#menubar {<br />
font-weight: bold;<br />
font-size: 13px;<br />
line-height: 20px;<br />
}<br />
<br />
div#menubar.right-menu {<br />
float: right;<br />
}<br />
<br />
div#menubar.left-menu {<br />
float: left;<br />
}<br />
<br />
/* Fix text */<br />
div#menubar.left-menu a, div#menubar.left-menu li {<br />
text-transform: capitalize;<br />
}<br />
<br />
div#menubar li {<br />
padding: 0px 5px;<br />
display: inline;<br />
}<br />
<br />
/* White on hover */<br />
div#menubar a:hover {<br />
color: #FFFFFF;<br />
text-decoration: none;<br />
}<br />
<br />
div#menubar li[style="color:white;cursor:default"] {<br />
display: none;<br />
}<br />
<br />
/* End top-section style ================================================ */<br />
<br />
<br />
/* Start header style =================================================== */<br />
<br />
/* Hide page title (it would appear before the header) */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
/* Header-bg and positioning */<br />
div#header-container {<br />
background: url('https://static.igem.org/mediawiki/2013/b/bf/TUM13_page-bg-top.png') no-repeat 50% 0%;<br />
height: 217px;<br />
padding-top: 30px;<br />
}<br />
<br />
/* Header positioning */<br />
div#header {<br />
width: 920px;<br />
height: 130px;<br />
margin: 0px auto;<br />
padding: 5px 20px 0px;<br />
overflow: visible;<br />
}<br />
<br />
/* Logos */<br />
div#header img#physco-logo {<br />
display: block;<br />
height: 130px;<br />
margin-left: 303px;<br />
}<br />
<br />
div#header img#igem-logo {<br />
display: block;<br />
margin-top: 40px;<br />
height: 95px;<br />
float: right;<br />
}<br />
<br />
div#header img#tum-logo {<br />
display: block;<br />
margin-top: 15px;<br />
height: 120px;<br />
float: left;<br />
}<br />
<br />
/* End header style ===================================================== */<br />
<br />
<br />
/* Start menu style ===================================================== */<br />
<br />
/* Menu-bg and spacing*/<br />
div#menu {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a8/TUM13_menu-bg.png') no-repeat 50% 0%;<br />
height: 60px;<br />
padding: 5px 0px 17px;<br />
}<br />
<br />
div#menu ul {<br />
list-style: none;<br />
padding: 0;<br />
margin: 0;<br />
}<br />
<br />
/* Position menu and set font color */<br />
div#menu > ul {<br />
margin: 0px auto;<br />
width: 960px;<br />
height: 60px;<br />
color: #411c03;<br />
}<br />
<br />
/* Position menu elements and set font */<br />
div#menu > ul > li {<br />
float: left;<br />
width: 137px;<br />
height: 44px;<br />
padding: 8px 0px 8px;<br />
text-align: center;<br />
position: relative;<br />
font: normal 24px/44px sketch_rockwell,Arial,sans-serif;<br />
}<br />
<br />
div#menu > ul > li > a {<br />
display: block;<br />
width: 137px;<br />
height: 44px;<br />
color: inherit;<br />
}<br />
<br />
/* Hover effect */<br />
div#menu > ul > li > a:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/9/95/TUM13_menu-highlight.png') repeat-x 50% 50%;<br />
}<br />
<br />
div#menu > ul > li > a:hover {<br />
text-decoration: none;<br />
}<br />
<br />
/* Style and hide submenu */<br />
div#menu > ul > li > ul {<br />
border-left: solid 1px #411c03;<br />
border-right: solid 1px #411c03;<br />
width: 135px;<br />
background: #7cac2c;<br />
display: none;<br />
position: absolute;<br />
top: 60px;<br />
z-index: 9998;<br />
}<br />
<br />
/* Style submenu elements */<br />
div#menu > ul > li > ul > li {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4e/TUM13_submenu-bg.png') repeat-y 50% 50%;<br />
padding: 0px 10px;<br />
text-align: left;<br />
border-bottom: solid 1px #411c03;<br />
font: bold 13px/20px Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
div#menu > ul > li > ul > li > a {<br />
display: block;<br />
width: 115px;<br />
padding: 5px 0px;<br />
color: inherit;<br />
}<br />
<br />
/* Hover effect */<br />
div#menu > ul > li > ul > li:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/db/TUM13_submenu-highlight.png') repeat-y 50% 50%;<br />
}<br />
<br />
div#menu > ul > li > ul > li > a:hover {<br />
text-decoration: none;<br />
}<br />
<br />
/* Show submenu on hover */<br />
div#menu > ul > li:hover > ul {<br />
display: block;<br />
}<br />
<br />
/* End menu style ======================================================= */<br />
<br />
<br />
/* Start content style ================================================== */<br />
<br />
div#bodyContent > p, div#contentSub, div#catlinks, table#toc.toc {<br />
display: none;<br />
}<br />
/*<br />
div#wikicontent > table#toc.toc {<br />
width: 350px;<br />
position: fixed;<br />
top: 100px;<br />
left: -300px;<br />
z-index: 10001;<br />
}<br />
<br />
<br />
div#wikicontent > table#toc.toc:hover {<br />
left: 0px;<br />
}<br />
*/<br />
/* top button */<br />
div#social-footer a#gotop {<br />
display: block;<br />
width: 40px;<br />
height: 40px;<br />
padding: 10px;<br />
background: url('https://static.igem.org/mediawiki/2013/2/2d/TUM13_gotop.png') no-repeat 50% 50%;<br />
position: fixed;<br />
bottom: 15px;<br />
right: 15px;<br />
}<br />
<br />
div#social-footer a#gotop:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c5/TUM13_gotop-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
/* Set page-bg */<br />
div#wikicontent-container {<br />
background: url('https://static.igem.org/mediawiki/2013/a/ac/TUM13_page-bg-middle.png') repeat-y 50% 0%;<br />
padding: 2px 0px 1px;<br />
}<br />
<br />
div#wikicontent > :first-child {<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent > :last-child {<br />
margin-bottom: 0px;<br />
}<br />
<br />
/* Font and position */<br />
div#wikicontent {<br />
font-size: 14px;<br />
line-height: 21px;<br />
background: #e9e3dd;<br />
width: 920px;<br />
margin: 0px auto;<br />
padding: 0px 20px;<br />
text-align: justify;<br />
position: relative;<br />
}<br />
<br />
/* Lower page border */<br />
div#bodyContent > div.visualClear {<br />
background: url('https://static.igem.org/mediawiki/2013/6/6e/TUM13_page-bg-bottom.png') no-repeat 50% 0%;<br />
height: 50px;<br />
}<br />
<br />
/* Standard elements */<br />
div#wikicontent p {<br />
margin: 0 0 1em;<br />
}<br />
<br />
div#wikicontent ul {<br />
padding-left: 2em;<br />
list-style: disc outside;<br />
}<br />
<br />
div#wikicontent div.achievements ul {<br />
list-style: none;<br />
padding-left: 0.4em;<br />
}<br />
<br />
div#wikicontent div.achievements ul li {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_checkmark-small.png') no-repeat 0% 50%;<br />
padding-left: 30px;<br />
margin: 0px;<br />
line-height: 32px;<br />
}<br />
<br />
div#wikicontent div.achievements h3, div#wikicontent div.achievements h4, div#wikicontent div.achievements h5, div#wikicontent div.achievements h6 {<br />
clear: none;<br />
}<br />
<br />
div#wikicontent ol {<br />
padding-left: 2em;<br />
list-style: decimal outside;<br />
}<br />
<br />
div#wikicontent li {<br />
margin-bottom: 0.5em;<br />
}<br />
<br />
div#wikicontent a {<br />
color: #7cac2c;<br />
}<br />
<br />
div#bodyContent a[href^="https://"], div#bodyContent a[href^="mailto:"], #bodyContent .link-https {<br />
background: transparent;<br />
padding: 0px;<br />
}<br />
<br />
div#wikicontent a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
/* Headings */<br />
div#wikicontent h1, div#wikicontent h2, div#wikicontent h3, div#wikicontent h4, div#wikicontent h5, div#wikicontent h6 {<br />
font-weight: bold;<br />
line-height: 1.5;<br />
margin: 1em 0em 0.5em;<br />
clear: both;<br />
text-align: left;<br />
}<br />
<br />
div#wikicontent .editsection {<br />
color: #7cac2c;<br />
font-family: Helvetica,Arial,Verdana,sans-serif;<br />
}<br />
<br />
div#wikicontent h1, div#wikicontent h2 {<br />
color: #502204;<br />
font: normal 2em/1.35 sketch_rockwell,Arial,sans-serif;<br />
border-bottom: 2px solid #502204;<br />
}<br />
<br />
div#wikicontent h3 {<br />
font-size: 1.5em;<br />
}<br />
<br />
div#wikicontent h4 {<br />
font-size: 1.25em;<br />
}<br />
<br />
div#wikicontent h5, div#wikicontent h6 {<br />
font-size: 1.0em;<br />
}<br />
<br />
/* Pictures */<br />
div#wikicontent div.thumb {<br />
border: none;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner {<br />
background: #ded6ca;<br />
border: none;<br />
font-size: 12px;<br />
line-height: 23px;<br />
overflow: hidden;<br />
padding: 5px 4px 2px !important;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner a img {<br />
border: none;<br />
padding-bottom: 3px;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption {<br />
background: transparent;<br />
font-size: inherit;<br />
line-height: inherit;<br />
overflow: hidden;<br />
padding: 0px 5px !important;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption div.magnify {<br />
display: none;<br />
}<br />
<br />
div#wikicontent div.tright {<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent div.tleft {<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
div#wikicontent div.center div.tnone {<br />
clear: both;<br />
margin: 0 0 1em;<br />
}<br />
<br />
/* Tables */<br />
div#wikicontent table {<br />
background: #ded6ca;<br />
border: 1px solid #502204;<br />
margin: 0 auto 1em;<br />
}<br />
<br />
div#wikicontent table.left {<br />
float: left;<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
<br />
div#wikicontent table.right {<br />
float: right;<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent table.safety td, div#wikicontent table.safety th {<br />
text-align: left;<br />
vertical-align: top;<br />
}<br />
<br />
div#wikicontent table caption {<br />
text-align: left;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent td, div#wikicontent th {<br />
border: 1px solid #502204;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#wikicontent th {<br />
background: #d4c6b6;<br />
font-weight: bold;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent table code {<br />
background: transparent;<br />
}<br />
<br />
/* Fix AutoAnnotator */<br />
div#wikicontent table#AutoAnnotator, div#wikicontent table#AutoAnnotator table {<br />
background: #ffffff;<br />
border: 1px solid #000000;<br />
font-size: 12px;<br />
line-height: 18px;<br />
margin-bottom: 0px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator td, div#wikicontent table#AutoAnnotator th {<br />
border: 1px solid #000000;<br />
padding: 1px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder th {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder th {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator th {<br />
background: rgb(221, 221, 221);<br />
}<br />
<br />
div#wikicontent div#autoannotator-computing {<br />
text-align: center;<br />
}<br />
<br />
/* Fix parts table */<br />
div#groupparts {<br />
width: 920px !important;<br />
}<br />
<br />
div#groupparts td {<br />
background: #ded6ca;<br />
}<br />
<br />
/* Sponsors */<br />
div#wikicontent a.sponsor {<br />
display: block;<br />
float: left;<br />
width: 286px;<br />
height: 100px;<br />
margin: 10px;<br />
background: #FFFFFF;<br />
position: relative;<br />
}<br />
<br />
div#wikicontent a.sponsor img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
bottom: 0;<br />
margin: auto;<br />
}<br />
<br />
div#wikicontent div.sponsor {<br />
background: #ded6ca;<br />
height: 215px;<br />
margin-bottom: 20px;<br />
padding: 0px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.sponsor a.sponsor {<br />
width: 320px;<br />
height: 215px;<br />
margin: 0 1em 0 0;<br />
}<br />
<br />
div#wikicontent div.sponsor p {<br />
padding: 1em;<br />
}<br />
<br />
div div#wikicontent div.overview {<br />
height: 240px;<br />
width: 440px;<br />
padding: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview img {<br />
float: right;<br />
margin: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview h3 {<br />
margin: 5px 0px 0px;<br />
clear: none;<br />
}<br />
<br />
div#wikicontent div.overview span.editsection {<br />
display: none;<br />
}<br />
<br />
/* Boxes */<br />
div#wikicontent div.box-right {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 0px 20px 10px;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
}<br />
<br />
div#wikicontent div.box-left {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 10px 20px 0px;<br />
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/* End content style ==================================================== */<br />
<br />
<br />
/* Start footer style =================================================== */<br />
<br />
/* Makes the footer stick to the bottom */<br />
html, body {<br />
height: 100%;<br />
} <br />
<br />
body > div#globalWrapper {<br />
position: relative;<br />
height: auto;<br />
min-height: 100%;<br />
margin: 0px auto -156px;<br />
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div#globalWrapper > div.visualClear {<br />
height: 156px;<br />
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background: url('https://static.igem.org/mediawiki/2013/c/c3/TUM13_footer-bg.png') repeat-x 50% 0%;<br />
padding: 5px 0px 10px;<br />
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position: absolute;<br />
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color: #d4c6b6;<br />
font-weight: normal;<br />
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<br />
div#social-footer {<br />
width: 940px;<br />
height: 95px;<br />
margin: 0px auto;<br />
padding: 0px 10px 10px;<br />
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div#social-footer div#annotator, div#social-footer div#address, div#social-footer div#follow {<br />
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height: 95px;<br />
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div#social-footer h2 {<br />
font: normal 28px/42px sketch_rockwell, Arial, sans-serif;<br />
text-align: center;<br />
margin: 0px;<br />
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text-align: center;<br />
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div#social-footer div#annotator input {<br />
font-size: 12px;<br />
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div#social-footer div#annotator input[type="text"] {<br />
margin: 0px 0px 5px;<br />
border: 1px solid #502204;<br />
background: #d4c6b6;<br />
width: 275px;<br />
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div#social-footer div#address p {<br />
margin: 0px;<br />
width: 50%;<br />
float: left;<br />
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<br />
div#social-footer div#follow a {<br />
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width: 32px;<br />
height: 32px;<br />
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div#social-footer div#follow div {<br />
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div#social-footer div#follow a#adress {<br />
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div#social-footer div#follow a#mail {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_contact-icon.png') no-repeat 50% 50%;<br />
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div#social-footer div#follow a#mail:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c7/TUM13_contact-icon-highlight.png') no-repeat 50% 50%;<br />
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text-align: center;<br />
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div#footer ul {<br />
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padding: 0;<br />
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div#footer li {<br />
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display: inline;<br />
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div#footer-box a {<br />
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<br />
div#footer div#f-copyrightico {<br />
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float: left;<br />
}<br />
<br />
/* End footer style ===================================================== */</div>FlorianAhttp://2013.igem.org/File:TUM13_checkmark-small.pngFile:TUM13 checkmark-small.png2014-05-09T18:45:47Z<p>FlorianA: uploaded a new version of &quot;File:TUM13 checkmark-small.png&quot;</p>
<hr />
<div></div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T18:40:37Z<p>FlorianA: /* Achievements */</p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
<br />
<div id="wikicontent-container"><br />
<div id="wikicontent"><html><ul class="bxslider"> <!-- das muss so hässlich sein, damit kein absatz eingefügt wird --><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/97/TUM13_slider_team1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/66/TUM13_slider_kampen.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/30/TUM13_slider_moos.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/TUM13_Foto_Germanmuseum_12.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a9/TUM13_labor.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/30/TUM13_slider_team2.jpg" /></li><br />
</ul><br />
<div style="height: 50px; margin: 14px 0px 5px;"><br />
<div style="float: left; text-align: right; width: 140px; height: 40px; margin: 5px 0px;"><br />
<span id="counter" style="color: #502204; font: normal 24px/40px sketch_rockwell,Arial,sans-serif;">0</span> <a href="https://2013.igem.org/Special:PopularPages">visitors</a><br />
</div><br />
<div style="float: left; text-align: right; width: 400px; height: 40px; margin: 5px 0px 5px 33px;"><br />
<span id="countdown" style="color: #502204; font: normal 24px/40px sketch_rockwell,Arial,sans-serif;"></span> left to World Wiki Freeze<br />
</div><br />
<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
</div><br />
<div style="height: 145px; margin: 5px 0px 5px;"><br />
<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
<a class="button safety" href="https://2013.igem.org/Team:TU-Munich/Project/Safety">Safety</a><br />
<a class="button judging" href="https://2013.igem.org/Team:TU-Munich/Team/Judging">Judging</a><br />
</div><br />
</html><br />
<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><div class="thumbinner" style="width:502px;"><a href="/File:TUM13_blubbern-stop.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="450" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer</div></div></div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
==== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Best Environment Project<br />
* World First Runner Up<br />
==== Business plan competitions after iGEM: ====<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
<html><br />
<a class="sponsor" href="http://www.tum.de/"><img src="https://static.igem.org/mediawiki/2013/9/97/Logo_TU-Muenchen_01.png" width="190px"></a><br />
<a class="sponsor" href="http://wzw.tum.de/"><img src="https://static.igem.org/mediawiki/2013/a/ac/Wzwlogo2.png" width="210px"></a><br />
<a class="sponsor" href="http://biologische-chemie.userweb.mwn.de/index.html"><img src="https://static.igem.org/mediawiki/2013/0/05/Unbenannt.PNG" width="190px"></a><br />
<a class="sponsor" href="http://www.daad.de/de/index.html"><img src="https://static.igem.org/mediawiki/2013/f/f7/TUM13_DAAD.jpg" width="120px"></a><br />
<a class="sponsor" href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="190px"></a><br />
<a class="sponsor" href="http://www.eurofins.de/"><img src="https://static.igem.org/mediawiki/2013/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a class="sponsor" href="http://www.mathworks.de/"><img src="https://static.igem.org/mediawiki/2013/0/0c/TUM13_MathWorks.png" width="200px"></a><br />
<a class="sponsor" href="http://www.ika.com/"><img src="https://static.igem.org/mediawiki/2013/9/9d/TUM13_IKA.png" width="105px"></a><br />
<a class="sponsor" href="http://www.neb.com/"><img src="https://static.igem.org/mediawiki/2013/5/50/TUM13_Sponsor_NEB.jpg" width="155px"></a><br />
<a class="sponsor" href="http://www.promega.de/"><img src="https://static.igem.org/mediawiki/2013/f/f0/Promega-300.jpg" width="175px"></a><br />
<a class="sponsor" href="http://www.qiagen.com/"><img src="https://static.igem.org/mediawiki/2013/6/60/TUM13_Sponsor_Qiagen.png" width="100px"></a> <br />
<a class="sponsor" href="http://www.iba-lifesciences.com/"><img src="https://static.igem.org/mediawiki/2013/9/9c/Iba_logo_claim_r_farbe.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://eu.idtdna.com/site/"><img src="https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://www.thermoscientific.com"><img src="https://static.igem.org/mediawiki/2013/e/ed/TUM13_Sponsor_logo_Thermo.jpg" width="140px"></a><br />
<a class="sponsor" href="http://www.erasynbio.eu/"><img src="https://static.igem.org/mediawiki/2013/5/58/Logo_ERASynBio.png" width="180px"></a><br />
<div class="visualClear"></div><br />
<a class="sponsor" style="float: none; margin: 10px auto;" href="http://www.roche.com/"><img src="https://static.igem.org/mediawiki/2013/d/d2/TUM13_Roche.jpg" height="80px"></a><br />
<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T18:38:31Z<p>FlorianA: </p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
<br />
<div id="wikicontent-container"><br />
<div id="wikicontent"><html><ul class="bxslider"> <!-- das muss so hässlich sein, damit kein absatz eingefügt wird --><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/97/TUM13_slider_team1.jpg" /></li><br />
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</ul><br />
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<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
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<div style="height: 145px; margin: 5px 0px 5px;"><br />
<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
<a class="button safety" href="https://2013.igem.org/Team:TU-Munich/Project/Safety">Safety</a><br />
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<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><div class="thumbinner" style="width:502px;"><a href="/File:TUM13_blubbern-stop.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="450" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer</div></div></div></html><br />
<div class="achievements"><br />
==== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ====<br />
* Gold medal<br />
* Best Wiki<br />
* European First Runner Up<br />
=== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ===<br />
* Best Environment Project<br />
* World First Runner Up<br />
=== Business plan competitions after iGEM: ===<br />
* 1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
* 2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project competition] <br />
* Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
</div><br />
<br />
== Sponsors ==<br />
<html><br />
<a class="sponsor" href="http://www.tum.de/"><img src="https://static.igem.org/mediawiki/2013/9/97/Logo_TU-Muenchen_01.png" width="190px"></a><br />
<a class="sponsor" href="http://wzw.tum.de/"><img src="https://static.igem.org/mediawiki/2013/a/ac/Wzwlogo2.png" width="210px"></a><br />
<a class="sponsor" href="http://biologische-chemie.userweb.mwn.de/index.html"><img src="https://static.igem.org/mediawiki/2013/0/05/Unbenannt.PNG" width="190px"></a><br />
<a class="sponsor" href="http://www.daad.de/de/index.html"><img src="https://static.igem.org/mediawiki/2013/f/f7/TUM13_DAAD.jpg" width="120px"></a><br />
<a class="sponsor" href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="190px"></a><br />
<a class="sponsor" href="http://www.eurofins.de/"><img src="https://static.igem.org/mediawiki/2013/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a class="sponsor" href="http://www.mathworks.de/"><img src="https://static.igem.org/mediawiki/2013/0/0c/TUM13_MathWorks.png" width="200px"></a><br />
<a class="sponsor" href="http://www.ika.com/"><img src="https://static.igem.org/mediawiki/2013/9/9d/TUM13_IKA.png" width="105px"></a><br />
<a class="sponsor" href="http://www.neb.com/"><img src="https://static.igem.org/mediawiki/2013/5/50/TUM13_Sponsor_NEB.jpg" width="155px"></a><br />
<a class="sponsor" href="http://www.promega.de/"><img src="https://static.igem.org/mediawiki/2013/f/f0/Promega-300.jpg" width="175px"></a><br />
<a class="sponsor" href="http://www.qiagen.com/"><img src="https://static.igem.org/mediawiki/2013/6/60/TUM13_Sponsor_Qiagen.png" width="100px"></a> <br />
<a class="sponsor" href="http://www.iba-lifesciences.com/"><img src="https://static.igem.org/mediawiki/2013/9/9c/Iba_logo_claim_r_farbe.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://eu.idtdna.com/site/"><img src="https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg" width="180px"></a> <br />
<a class="sponsor" href="http://www.thermoscientific.com"><img src="https://static.igem.org/mediawiki/2013/e/ed/TUM13_Sponsor_logo_Thermo.jpg" width="140px"></a><br />
<a class="sponsor" href="http://www.erasynbio.eu/"><img src="https://static.igem.org/mediawiki/2013/5/58/Logo_ERASynBio.png" width="180px"></a><br />
<div class="visualClear"></div><br />
<a class="sponsor" style="float: none; margin: 10px auto;" href="http://www.roche.com/"><img src="https://static.igem.org/mediawiki/2013/d/d2/TUM13_Roche.jpg" height="80px"></a><br />
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<center style="margin-top: 10px;"><a href="http://www2.clustrmaps.com/user/c81109e0d" id="clustrMapsLink"><img src="http://www2.clustrmaps.com/stats/maps-no_clusters/2013.igem.org-Team-TU-Munich-thumb.jpg" 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';" width="150" /></a></center><br />
</html><br />
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<!-- Ende des Inhalts --><br />
</div><br />
</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/TUM13_wiki.cssTeam:TU-Munich/TUM13 wiki.css2014-05-09T18:38:27Z<p>FlorianA: </p>
<hr />
<div>/* iGEM TU-Munich Stylesheet by Florian Albrecht (albrecht.flo@gmx.net)<br />
if you want to use parts of this on your wiki, cite us and / or write an e-mail to us */<br />
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/* Logos */<br />
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div#wikicontent h3 {<br />
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}<br />
<br />
div#wikicontent h4 {<br />
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}<br />
<br />
div#wikicontent h5, div#wikicontent h6 {<br />
font-size: 1.0em;<br />
}<br />
<br />
/* Pictures */<br />
div#wikicontent div.thumb {<br />
border: none;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner {<br />
background: #ded6ca;<br />
border: none;<br />
font-size: 12px;<br />
line-height: 23px;<br />
overflow: hidden;<br />
padding: 5px 4px 2px !important;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner a img {<br />
border: none;<br />
padding-bottom: 3px;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption {<br />
background: transparent;<br />
font-size: inherit;<br />
line-height: inherit;<br />
overflow: hidden;<br />
padding: 0px 5px !important;<br />
}<br />
<br />
div#wikicontent div.thumb div.thumbinner div.thumbcaption div.magnify {<br />
display: none;<br />
}<br />
<br />
div#wikicontent div.tright {<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent div.tleft {<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
div#wikicontent div.center div.tnone {<br />
clear: both;<br />
margin: 0 0 1em;<br />
}<br />
<br />
/* Tables */<br />
div#wikicontent table {<br />
background: #ded6ca;<br />
border: 1px solid #502204;<br />
margin: 0 auto 1em;<br />
}<br />
<br />
div#wikicontent table.left {<br />
float: left;<br />
clear: left;<br />
margin: 0 1em 1em 0;<br />
}<br />
<br />
<br />
div#wikicontent table.right {<br />
float: right;<br />
clear: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#wikicontent table.safety td, div#wikicontent table.safety th {<br />
text-align: left;<br />
vertical-align: top;<br />
}<br />
<br />
div#wikicontent table caption {<br />
text-align: left;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent td, div#wikicontent th {<br />
border: 1px solid #502204;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#wikicontent th {<br />
background: #d4c6b6;<br />
font-weight: bold;<br />
text-align: center;<br />
}<br />
<br />
div#wikicontent table code {<br />
background: transparent;<br />
}<br />
<br />
/* Fix AutoAnnotator */<br />
div#wikicontent table#AutoAnnotator, div#wikicontent table#AutoAnnotator table {<br />
background: #ffffff;<br />
border: 1px solid #000000;<br />
font-size: 12px;<br />
line-height: 18px;<br />
margin-bottom: 0px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator td, div#wikicontent table#AutoAnnotator th {<br />
border: 1px solid #000000;<br />
padding: 1px;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorNoBorder th {<br />
border: none;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder td, div#wikicontent table#AutoAnnotator table.AutoAnnotatorWithBorder th {<br />
border: 1px solid #000000;<br />
}<br />
<br />
div#wikicontent table#AutoAnnotator th {<br />
background: rgb(221, 221, 221);<br />
}<br />
<br />
div#wikicontent div#autoannotator-computing {<br />
text-align: center;<br />
}<br />
<br />
/* Fix parts table */<br />
div#groupparts {<br />
width: 920px !important;<br />
}<br />
<br />
div#groupparts td {<br />
background: #ded6ca;<br />
}<br />
<br />
/* Sponsors */<br />
div#wikicontent a.sponsor {<br />
display: block;<br />
float: left;<br />
width: 286px;<br />
height: 100px;<br />
margin: 10px;<br />
background: #FFFFFF;<br />
position: relative;<br />
}<br />
<br />
div#wikicontent a.sponsor img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
bottom: 0;<br />
margin: auto;<br />
}<br />
<br />
div#wikicontent div.sponsor {<br />
background: #ded6ca;<br />
height: 215px;<br />
margin-bottom: 20px;<br />
padding: 0px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.sponsor a.sponsor {<br />
width: 320px;<br />
height: 215px;<br />
margin: 0 1em 0 0;<br />
}<br />
<br />
div#wikicontent div.sponsor p {<br />
padding: 1em;<br />
}<br />
<br />
div div#wikicontent div.overview {<br />
height: 240px;<br />
width: 440px;<br />
padding: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview img {<br />
float: right;<br />
margin: 0px 0px 0px 10px;<br />
}<br />
<br />
div#wikicontent div.overview h3 {<br />
margin: 5px 0px 0px;<br />
clear: none;<br />
}<br />
<br />
div#wikicontent div.overview span.editsection {<br />
display: none;<br />
}<br />
<br />
/* Boxes */<br />
div#wikicontent div.box-right {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 0px 20px 10px;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
}<br />
<br />
div#wikicontent div.box-left {<br />
width: 430px;<br />
background: #ded6ca;<br />
margin: 0px 10px 20px 0px;<br />
padding: 10px 10px 0px;<br />
clear: left;<br />
float: left;<br />
}<br />
<br />
div#wikicontent div.team.box-left, div#wikicontent div.team.box-right {<br />
height: 620px;<br />
}<br />
<br />
div#wikicontent div.team div, div#wikicontent div.team a img {<br />
float: right;<br />
position: relative;<br />
margin: 0 0 10px 10px;<br />
background: url('https://static.igem.org/mediawiki/2013/4/4d/TUM13_computing.gif') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div.team div.portrait {<br />
width: 200px;<br />
height: 356px;<br />
}<br />
<br />
div#wikicontent div.team div.landscape {<br />
width: 300px;<br />
height: 169px;<br />
}<br />
<br />
div#wikicontent div.team div img {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
}<br />
<br />
div#wikicontent div.team div img.top {<br />
z-index: 20;<br />
}<br />
<br />
div#wikicontent div.team div:hover img.top {<br />
visibility: hidden;<br />
}<br />
<br />
div#wikicontent div.team div img.bottom {<br />
z-index: 10;<br />
}<br />
<br />
div#wikicontent div.box-definition {<br />
width: 320px;<br />
background: #ded6ca;<br />
margin: 0 0 1em 1em;<br />
padding: 10px 10px 0px;<br />
clear: right;<br />
float: right;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#wikicontent div.box-definition p {<br />
margin-bottom: 10px;<br />
}<br />
<br />
div#wikicontent div.box-center {<br />
background: #ded6ca;<br />
margin-bottom: 20px;<br />
padding: 10px;<br />
clear: both;<br />
}<br />
<br />
div#wikicontent div.team h4 {<br />
margin-top: 0px;<br />
}<br />
<br />
div#wikicontent div.quote {<br />
margin: 1em 1.5em;<br />
padding: 0.5em 0.7em;<br />
background: #ded6ca;<br />
}<br />
<br />
div#wikicontent div.quote p {<br />
margin: 0px;<br />
font: normal 20px/30px Garamond, Baskerville, "Baskerville Old Face", "Hoefler Text", "Times New Roman", serif;<br />
/* font-style: italic; */<br />
}<br />
<br />
div#wikicontent div.quote div.author {<br />
text-align: right;<br />
font-variant: small-caps;<br />
}<br />
<br />
/* Buttons */<br />
<br />
div#wikicontent a.tour {<br />
display: block;<br />
font: normal 24px/40px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.tour.start {<br />
float: right;<br />
width: 205px;<br />
height: 40px;<br />
padding: 5px 7px 5px 38px;<br />
background: url('https://static.igem.org/mediawiki/2013/3/36/TUM13_tour-start.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.start:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/5/5f/TUM13_tour-start-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous {<br />
float: left;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 5px 5px 25px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/f/fc/TUM13_tour-previous.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.previous:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/6/6e/TUM13_tour-previous-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next {<br />
float: right;<br />
width: 135px;<br />
height: 40px;<br />
padding: 5px 25px 5px 5px;<br />
margin-bottom: 20px;<br />
background: url('https://static.igem.org/mediawiki/2013/c/c6/TUM13_tour-next.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.tour.next:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/3/35/TUM13_tour-next-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button {<br />
display: block;<br />
float: left;<br />
width: 131px;<br />
height: 20px;<br />
padding: 11px 7px 114px;<br />
margin-left: 10px;<br />
font: normal 16px/20px sketch_rockwell,Arial,sans-serif;<br />
color: #411c03;<br />
text-align: center;<br />
text-decoration: none;<br />
}<br />
<br />
div#wikicontent a.button.application {<br />
margin-left: 0px;<br />
background: url('https://static.igem.org/mediawiki/2013/7/7b/TUM13_button-application.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.application:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/4/44/TUM13_button-application-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator {<br />
background: url('https://static.igem.org/mediawiki/2013/4/43/TUM13_button-autoannotator.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.autoannotator:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a7/TUM13_button-autoannotator-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco {<br />
background: url('https://static.igem.org/mediawiki/2013/7/77/TUM13_button-physco.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.physco:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d5/TUM13_button-physco-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e1/TUM13_button-entrepreneur.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.entrepreneur:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/e/e6/TUM13_button-entrepreneur-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4f/TUM13_button-safety.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.safety:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/01/TUM13_button-safety-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging {<br />
background: url('https://static.igem.org/mediawiki/2013/4/47/TUM13_button-judging.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent a.button.judging:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d6/TUM13_button-judging-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay {<br />
width: 100%;<br />
height: 100%;<br />
z-index: 9996;<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
background: url('https://static.igem.org/mediawiki/2013/2/29/TUM13_ajax-bg.png') repeat 0% 0%;<br />
}<br />
<br />
div#wikicontent div#ajax-overlay img {<br />
position: absolute;<br />
z-index: 9997;<br />
top: 0;<br />
left: 0;<br />
right: 0;<br />
margin: auto;<br />
}<br />
<br />
/* End content style ==================================================== */<br />
<br />
<br />
/* Start footer style =================================================== */<br />
<br />
/* Makes the footer stick to the bottom */<br />
html, body {<br />
height: 100%;<br />
} <br />
<br />
body > div#globalWrapper {<br />
position: relative;<br />
height: auto;<br />
min-height: 100%;<br />
margin: 0px auto -156px;<br />
}<br />
<br />
div#globalWrapper > div.visualClear {<br />
height: 156px;<br />
}<br />
<br />
div#footer-box {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c3/TUM13_footer-bg.png') repeat-x 50% 0%;<br />
padding: 5px 0px 10px;<br />
height: 141px;<br />
width: 100%;<br />
position: absolute;<br />
bottom: 0px;<br />
color: #d4c6b6;<br />
font-weight: normal;<br />
font-size: 12px;<br />
line-height: 18px;<br />
}<br />
<br />
div#social-footer {<br />
width: 940px;<br />
height: 95px;<br />
margin: 0px auto;<br />
padding: 0px 10px 10px;<br />
}<br />
<br />
div#social-footer div#annotator, div#social-footer div#address, div#social-footer div#follow {<br />
width: 293px;<br />
height: 95px;<br />
margin: 0px 10px;<br />
float: left;<br />
}<br />
<br />
div#social-footer h2 {<br />
font: normal 28px/42px sketch_rockwell, Arial, sans-serif;<br />
text-align: center;<br />
margin: 0px;<br />
}<br />
<br />
div#social-footer div#annotator {<br />
text-align: center;<br />
}<br />
<br />
div#social-footer div#annotator input {<br />
font-size: 12px;<br />
padding: 2px 5px;<br />
}<br />
<br />
div#social-footer div#annotator input[type="text"] {<br />
margin: 0px 0px 5px;<br />
border: 1px solid #502204;<br />
background: #d4c6b6;<br />
width: 275px;<br />
}<br />
<br />
div#social-footer div#address p {<br />
margin: 0px;<br />
width: 50%;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow a {<br />
display: block;<br />
width: 32px;<br />
height: 32px;<br />
margin: 9px;<br />
float: left;<br />
}<br />
<br />
div#social-footer div#follow div {<br />
margin: 3px 21px 0px;<br />
}<br />
<br />
div#social-footer div#follow a#adress {<br />
background: url('https://static.igem.org/mediawiki/2013/4/4b/TUM13_address-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#adress:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/0/05/TUM13_address-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail {<br />
background: url('https://static.igem.org/mediawiki/2013/d/d8/TUM13_contact-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#mail:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/c/c7/TUM13_contact-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook {<br />
background: url('https://static.igem.org/mediawiki/2013/c/cb/TUM13_facebook-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#facebook:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/9/9f/TUM13_facebook-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter {<br />
background: url('https://static.igem.org/mediawiki/2013/3/34/TUM13_twitter-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#twitter:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/2/22/TUM13_twitter-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube {<br />
background: url('https://static.igem.org/mediawiki/2013/4/49/TUM13_youtube-icon.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#social-footer div#follow a#youtube:hover {<br />
background: url('https://static.igem.org/mediawiki/2013/a/a1/TUM13_youtube-icon-highlight.png') no-repeat 50% 50%;<br />
}<br />
<br />
div#footer {<br />
width: 920px;<br />
margin: 0px auto;<br />
padding: 0px 20px;<br />
text-align: center;<br />
}<br />
<br />
div#footer ul {<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
div#footer li {<br />
padding: 0px 5px;<br />
display: inline;<br />
}<br />
<br />
div#footer li#t-recentchangeslinked {<br />
display: none;<br />
}<br />
<br />
div#footer-box a {<br />
color: inherit;<br />
}<br />
<br />
div#footer-box a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
div#footer div#f-poweredbyico {<br />
margin-top: 3px;<br />
float: right;<br />
}<br />
<br />
div#footer div#f-copyrightico {<br />
margin-top: 3px;<br />
float: left;<br />
}<br />
<br />
/* End footer style ===================================================== */</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T18:32:34Z<p>FlorianA: </p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
<br />
<div id="wikicontent-container"><br />
<div id="wikicontent"><html><ul class="bxslider"> <!-- das muss so hässlich sein, damit kein absatz eingefügt wird --><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/97/TUM13_slider_team1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/66/TUM13_slider_kampen.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/30/TUM13_slider_moos.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/TUM13_Foto_Germanmuseum_12.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a9/TUM13_labor.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/30/TUM13_slider_team2.jpg" /></li><br />
</ul><br />
<div style="height: 50px; margin: 14px 0px 5px;"><br />
<div style="float: left; text-align: right; width: 140px; height: 40px; margin: 5px 0px;"><br />
<span id="counter" style="color: #502204; font: normal 24px/40px sketch_rockwell,Arial,sans-serif;">0</span> <a href="https://2013.igem.org/Special:PopularPages">visitors</a><br />
</div><br />
<div style="float: left; text-align: right; width: 400px; height: 40px; margin: 5px 0px 5px 33px;"><br />
<span id="countdown" style="color: #502204; font: normal 24px/40px sketch_rockwell,Arial,sans-serif;"></span> left to World Wiki Freeze<br />
</div><br />
<a class="tour start" href="https://2013.igem.org/Team:TU-Munich/Project/Overview">Take the Tour</a><br />
</div><br />
<div style="height: 145px; margin: 5px 0px 5px;"><br />
<a class="button application" href="https://2013.igem.org/Team:TU-Munich/Results/Implementation">Application</a><br />
<a class="button autoannotator" href="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">AutoAnnotator</a><br />
<a class="button entrepreneur" href="https://2013.igem.org/Team:TU-Munich/Results/Economics">Entrepreneur</a><br />
<a class="button physco" href="https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella">Physco</a><br />
<a class="button safety" href="https://2013.igem.org/Team:TU-Munich/Project/Safety">Safety</a><br />
<a class="button judging" href="https://2013.igem.org/Team:TU-Munich/Team/Judging">Judging</a><br />
</div><br />
</html><br />
<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
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== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><div class="thumbinner" style="width:402px;"><a href="/File:TUM13_blubbern-stop.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="400" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer</div></div></div></html><br />
<div class="achievements"><br />
=== European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ===<br />
*Gold medal<br />
*Best Wiki<br />
*European First Runner Up<br />
=== World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]: ===<br />
*Best Environment Project<br />
*World First Runner Up<br />
=== Business plan competitions after iGEM: ===<br />
*1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
*2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project competition] <br />
*Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2014-05-09T18:30:10Z<p>FlorianA: </p>
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<br />
== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
<br />
<html><br />
<right><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/76195786" width="900" height="510" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></right></html><br />
<br />
== Achievements ==<br />
<html><div class="thumb tright" id="blubberkolben"><div class="thumbinner" style="width:402px;"><a href="/File:TUM13_blubbern-stop.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2013/a/ab/TUM13_blubbern-stop.gif" width="400" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:TUM13_Physco-lifecycle.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Hover over the picture! Then you can actually HEAR the sound in our laboratory during the whole summer</div></div></div></html><br />
<div class="achievements"><br />
'''European Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]:'''<br />
*Gold medal<br />
*Best Wiki<br />
*European First Runner Up<br />
'''World Jamboree [https://igem.org/Results?year=2013&region=All&division=igem results]:'''<br />
*Best Environment Project<br />
*World First Runner Up<br />
'''Business plan competitions after iGEM:'''<br />
*1<sup>st</sup> place at the [http://www.tum.de/die-tum/aktuelles/pressemitteilungen/kurz/article/31350/ TUM IdeAward]<br />
*2<sup>nd</sup> place at the [http://www.presseportal.de/print/2731699-projekt-zum-internet-der-dinge-gewinnt-hochschulwettbewerb-der.html Be.Project competition] <br />
*Finalist in the second round of the [http://www.evobis.de/evobis/startupdemonight/nominierte_stufe_2_des_muenchener_businessplan_wettbewerbs_it_biotech_und_technologien/ Munich Business Plan Competition]<br />
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== Sponsors ==<br />
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<a class="sponsor" href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="190px"></a><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/File:TUM13_fabian-anim.gifFile:TUM13 fabian-anim.gif2013-10-29T03:59:00Z<p>FlorianA: uploaded a new version of &quot;File:TUM13 fabian-anim.gif&quot;: Reverted to version as of 01:51, 4 October 2013</p>
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<div></div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Team/CollaborationsTeam:TU-Munich/Team/Collaborations2013-10-29T03:42:56Z<p>FlorianA: /* Collaboration with Op.N */</p>
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==Collaboration with Dundee iGEM team 2013==<br />
<br />
[[File:TUM13_Collaboration_Dundee.png|thumb|right|350px|'''Figure 1:''' Shipment from the Dundee 2013 iGEM team.]]<br />
The [https://2013.igem.org/Team:Dundee Dundee] iGEM team 2013 also works on bioremediation: The toxin microcystin is released into water from lysed cyanobacteria and appears in great amounts during algal blooms. This cyclic peptide toxin covalently binds the protein phosphatase type 1 (PP1) and is thereby toxic for mammals. The idea of the Dundee iGEM team is to express the PP1 protein as an absorber for microcystin. We received their PP1 BioBrick, converted it from <nowiki>RFC 10 to RFC 25</nowiki> and constructed some expression plasmids to transform ''Physcomitrella patens''. Our purpose was applying Dundee's molecular mop in an aquatic, photoautotrophic chassis and thus expand their project's to a wider range. We successfully obtained equipped the moss plants with our synthetic moss receptor and extracellular PP1. '''Please visit our [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PhyscoFilter] results page to read more about this great collaboration.'''<br><br />
We'd also like to congratulate the Dundee iGEM team 2013 for their success at the European Jamboree: '''Well done Scots!'''<br />
<br />
==Collaboration with Glowing Plants==<br />
[[File:TUM13_Glowing_idea.png|thumb|left|300px|'''Figure 2:''' Outline of idea to utilise transgenic moss as ambient light source.]]<br />
[[File:TUM13_Blubber_flasks.gif|thumb|right|700px|'''Figure 3:''' Bubbling flask with our moss. This is how the moss would circulate in the bubble column reactor.]]<br />
We contacted the [http://glowingplant.com/ Glowing Plant] Team which has set itself the goal to develop glowing transgenic ''A. thaliana'' plants using luciferase based on the work of [https://2010.igem.org/Team:Cambridge iGEM Cambridge 2010]. They were quite interested in our idea using transgenic moss plants as light source and we agreed on a meeting on October 25<sup>th</sup>. Ahead of the European Jamboree (October 7 <sup>th</sup> they have also posted our project video on their [http://glowingplant.com/ blog] in order to promote it. We would love to see glowing ''Physcomitrella'' plants in the future.<br />
<br />
Glowingplant performed an extremely successful kickstarter campaign to raise money for their idea of plants which gather energy by photosynthesis and emit it again later by bioluminescence.<br />
<br />
On the 25<sup>th</sup> of October we had the great chance to meet [http://glowingplant.com/team Antony Evans] of the [http://glowingplant.com/ glowingplant team] on his visit to Europe. After having talked about the current status of our projects we had a great knowledge exchange about legal regulations, the public opinion on transgenic plants in Europe and in the US as well as about the potential sizes of the market for transgenic plants for home use. We intend to intensify our collaboration in the fashion of testing their gen cassette used for the luciferase reaction in our moss. It would be interesting to check how the utilized promoters for A. thaliana work in our moss ''P. patens''. However we plan to build a new gene construct using our tested Actin 5 promotor from ''Physcomitrella''.<br />
<br />
Maybe one day our moss will be usable to enlighten our living environment in aerated lava lamps like the one shown in figure 2. Having explored the scientific opportunities of our moss for this purpose glowingplant could become an extremely valuable partner on the way to a commercial product.<br />
<br />
==Collaboration with Paris-Saclay iGEM team 2013==<br />
<br />
[[File:TUM13_Collaboration_Paris.jpg|thumb|right|left|350px|'''Figure 4:''' Screenshot from our Skype conference with the Paris Saclay team.]]<br />
The [https://2013.igem.org/Team:Paris_Saclay Paris_Saclay] iGEM team is working on the detection and degradation of [http://en.wikipedia.org/wiki/Polychlorinated_biphenyl polychlorinated biphenyl] (PCB) in the context of bioremediation. We arranged a Skype-meeting, presented our projects to each other and agreed to '''exchange''' some of our coded '''BioBricks'''. The goal of that exchange was to test the new BioBricks in different chassis.<br />
<br />
==Collaboration with Op.N==<br />
[[File:TUM13_Collaboration_OpN.jpg|thumb|left|350px|'''Figure 5:''' An architects vision of our remediation rafts]]<br />
When we wondered how a moss filter could be implemented, we came across the NY PARALLEL NETWORKS concept which was designed by Op.N (Ali Fard and Ghazal Jafari). [http://op-n.net/filter/office/About-Op-N Op.N's work] considers "critical junction of architecture, landscape, and urban processes". The design and research practice is based in Cambridge and Toronto. Ali Fard and Ghazal Jafari are researchers at Harvard Graduate School of Design. <br />
<br />
We got in touch via E-Mail and found Ali and Ghazal interested in our ideas and '''willing to allow''' us the '''usage''' of their '''image material''' used at the ONE PRIZE competition.<br />
<br />
==Visiting Freiburg iGEM team 2013==<br />
[[File:Muenchen-Freiburg-2013.jpg|thumb|left|350px|'''Figure 6:''' iGEM Teams Freiburg and Munich joint during our first visit]]<br />
[[File:TUM13_Freiburg.JPG|thumb|right|200px|'''Figure 7:''' Our second visit]]<br />
During our [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#Trips_to_Freiburg trip to Freiburg] on August30<sup>th</sup> we met the iGEM team of Freiburg 2013. Along with a guidance through their lab and the Signalhaus they organized a city tour for us. This day ended with a barbecue together with the iGEM team. At this point we would like to thank you for your hospitality!<br />
<br />
== Exchange of urgently needed BioBricks ==<br />
<br />
===LMU Munich iGEM team 2012===<br />
We received the fluorescent proteins GFP, mKate2 and mVenus in RFC 25 from the 2012 Team of LMU Munich.<br />
<br><br />
===Tuebingen iGEM team 2013===<br />
We sent our pTUM100 vector system ([http://parts.igem.org/Part:BBa_K801000 BBa_K801000]) from the 2012 competition to the iGEM Team of Tuebingen.<br />
<br><br />
===Uppsala iGEM team 2013===<br />
We provided the iGEM Team Uppsala with CHS consless ([http://parts.igem.org/Part:BBa_K801095 BBa_K801095]), 4CL consless ([http://parts.igem.org/Part:BBa_K801093 BBa_K801093]) and PAL consless ([http://parts.igem.org/Part:BBa_K801091 BBa_K801091]) from the 2012 competition.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/HumanPractice/EducationTeam:TU-Munich/HumanPractice/Education2013-10-29T03:40:31Z<p>FlorianA: /* Experiment Number 1: Bacteria in Red */</p>
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== Introducing Synthetic Biology Kits at Educational Institutions ==<br />
<br />
What is better than teaching one class about SynBio?<br><br />
Enabling many teachers to complete this task!<br />
<br />
<div class="quote"><br />
"If you want to build a ship, do not drum up people together to collect wood and do not assign them tasks and work, but rather teach them to long for the endless immensity of the sea."<br />
<div class="author">- Antoine de Saint-Exupéry</div><br />
</div><br />
<br />
==Introduction==<br />
[[File:TUM13_Schulkits.jpg|thumb|right|400px|'''Figure 1:''' Prepared Educational-Kits, ready for delivery!]]<br />
Following this famous statement in pedagogics, we realized the importance to inspire and teach students in educational institutions to long for the endless immenseness of Synthetic Biology. We want to make it possible for disciples to take their first steps in the enormously evolving field of Synthetic Biology and let them take a breath of this subject, which will change the world in the near future due to its unlimited fields of application.<br />
<br />
The idea is to send an "Educational-Kit" with all required reagents and lab-protocols to educational institutions. Based on the experiments we designed we'd like to inspire young prospective people to think about a promising career in science. Our experiments are styled constitutive, based on every previously performed experiment, so every student, independent of training level, is able to understand and take notice of the resulting phenomena enabled by Synthetic Biology.<br />
<br />
==The Educational-Kit==<br />
The reagents, which are necessary for the realization of the experiments are all found in the kit, except for competent cells, which have to be stored at -20°C. It was set much value on the autonomous preparation of the final reagents, so nearly all reagents will be sent in their blank shape. Our aim is teaching the students all, or at least most, of the necessary steps of the designed experiments during the execution. The kit consists of the following reagents:<br />
<br />
{|cellspacing="0" border="1" right <br />
|+ '''Table 1:''' Contents of the educational kit<br />
! align="center";| Reagent<br />
! align="center";| Amount of the Reagent<br />
! align="center";| Is it existent in the kit?<br />
|-<br />
|LB-Medium<br />
| align=right | Reagents for about 3 L are in the kit<br />
| align=center | Yes<br />
|-<br />
|"Banana-Odor"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|"RFP-Generator"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|"Luciferase"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|Isoamylalcohol (>98 %)<br />
| align=right | 10 ml<br />
| align=center | Yes<br />
|-<br />
|Agar plates (Resistance: Kanamycin)<br />
| align=right | 4 Plates<br />
| align=center | Yes<br />
|-<br />
|Agar plates (Resistance: Chloramphenicol)<br />
| align=right | 2<br />
| align=center | Yes<br />
|-<br />
|L-Arabinose (500 mM)<br />
| align=right | 3.76 g (50 ml)<br />
| align=center | Yes<br />
|-<br />
|Kanamycin<br />
| align=right | 25 ml<br />
| align=center | Yes<br />
|-<br />
|Chloramphenicol<br />
| align=right | 25 ml<br />
| align=center | Yes<br />
|-<br />
|Competent Cells <br />
| align=right | 6 Tubes (Aliquotvolume: 150 µl)<br />
| align=center | No<br />
|-<br />
|} <br />
<br />
As you may have noticed, we provide three different plasmids, which are used separately in each designed experiment. After the experiments have been performed, disciples should have experienced the whole way from transformation, over cultivating cells, inducing the production of proteins. In the end they should also have observed the conversion of a substrate by the produced enzyme.<br />
<br />
==Experiment Number 1: Bacteria in Red==<br />
[[File:TUM13_RFP_Generator.jpg|thumb|right|400px|'''Figure 2:''' Results of Experiment Number 1]]<br />
The aim of the first experiment is to transform the [http://parts.igem.org/Part:BBa_K801100 "RFP-Generator"-Plasmid] into ''Escherichia coli'', which contains genes, coding for Chloramphenicol resistance, plus a red fluorophor. Then they should be plated on Chloramphenicol-Agarplates. The Agarplates, provided with the antibiotica Chloramphenicol, trigger a selection process, whose aim is to kill all bacteria which have not incorporated the "RFP-Generator"-Plasmid. The expression rate of the red fluorophor is controlled by a constitutive promoter, so the '''R'''ed '''F'''luorescent '''P'''rotein is expressed continuously. After just about one day of incubation time, the students can notice a change in color of the bacteria from ocher to red with the naked eye.<br />
<br />
The Biobrick we used to realize this experiment was firstly designed by the [https://2010.igem.org/Team:Groningen iGEM Team of Groningen in 2010] to simplify the cloning step by detecting internalized plasmids, which self-circularized without integration of the desired insert. Bacteria, which internalize self-circularized Plasmids will produce red colonies, so these undesired bacterial colonies can be avoided in the next steps of the experiment, to be sure of using the right construct.<br />
<br />
==Experiment Number 2: Glowing Bacteria Lamp==<br />
[[File:TUM13_Luciferase.jpg|thumb|right|300px|'''Figure 3:''' Structure of the Luciferase]]<br />
In the second experiment students should perform the transformation, which they learned in experiment one, with the "Luciferase"-Plasmid. The Plasmid encodes genes for Kanamycin resistance and a lux - gene cassette containing a protein-fluorophor called Luciferase. Another very important point is that the lux-gene is subordinated to a L-Arabinose Operon. After the transformation of the DNA-Plasmid has taken place, students should learn how to cultivate bacteria in liquid LB-Medium and how to induce protein expression by using L-Arabinose as an inductor of the L-Arabinose promoter. The glow of the Luciferase can already be observed after a few hours.<br />
<br />
==Experiment Number 3: Banana Odor Generator==<br />
[[File:SchmolkerMollath.png|thumb|right|400px|'''Figure 4:''' Results of Experiment three]]<br />
In the third experiment, all aspects learned in the previous experiments come together. The [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] encodes an enzyme called Alcohol-Acetyltransferase I. This enzyme is able to convert Isoamylalcohol into Isoamylacetat, which spreads the odor of banana. Additionally the plasmid harbors a resistance gene for Kanamycin and an inducible L-Arabinose promoter, which controls the expressions rate of the Alcohol-Acetyltransferase I enzyme. The students should transform the [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] into ''Escherichia coli'', cultivate the successfully transformed bacteria in a larger scale and in the end, induce the protein expression by using L-Arabinose. In the last step, the students should add the substrate Isoamylalcohol to the cell culture, so the produced enzyme can transform it into Isoamylacetat. <br />
<br />
The Biobrick, which spreads the odor of banana, after transforming its substrate, was first established by the [http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team of the Massachusetts Institute of Technology in 2006]. Their aim was to produce different compounds in ''Escherichia coli'' that smell fragrant. Another very interesting application of the "Banana-Odor" Biobrick is pursued by the [https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team of Queens in 2013]. This team tries to neutralize foot odor by creating a skin creme containing bacteria with a genetically engineered metabolic pathway to neutralize the volatile compounds that cause these smells. This pathway begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present in foot odor, and converts it into banana smell. By establishing this skin creme, it should be possible to prevent people from being attacked by mosquitos and saved from typical alienable diseases mosquitos spread.<br />
<br />
==Target Audience==<br />
The target audience for our kit are secondary schools with focus on a biotechnological educational pathway, so all students have access to the required laboratory equipment for the experiments. Before sending the kits to the schools, we tested them in detail - with success. All schools we contacted to spread our Synthetic Biology kits are very enthusiastic concerning our idea of supporting young prospective scientists, so they are very excited to receive and use our kit!<br />
<br />
==Availability of the School-kit==<br />
We appreciate other interested schools in trying out our “School kits”. Do not hesitate contacting us. We are happy to supply you with our forward-looking Synthetic Biology “Educational-kit”!<br />
<br />
You can '''send requests''' to: igem@wzw.tum.de<br />
<br />
Due to the World Championship Jamboree of iGEM, we offer the description of our "Educational Kit" directly to everyone, who is interested in it! Just click on the download-Button to get it! This decision supports our open-source idea, to facilitate the education of every pupil, prospective scientist and scientists. <br />
<br />
[[media:Educational_kit.pdf|'''Get your Educational kit!''']]<br />
<br />
We are very sad to tell you that our "Educational Kit" is just available in german because our target audience have been german secondary schools with a focus on biotechnological educational pathway. <br />
<br />
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==References==<br />
<br />
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[[https://2010.igem.org/Team:Groningen iGEM Team Groningen 2010]] <br><br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22368493 Close D et al., 2012]] Close D, Xu T, Smartt A, Rogers A, Crossley R, Price S, Ripp S, Sayler G (2012). The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter. ''Sensors'', 12(1):732-52. <br><br />
<br />
[[http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team Massachusetts Institute of Technology 2006]] <br><br />
<br />
[[https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team Queens 2013]] <br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/HumanPractice/InterviewsTeam:TU-Munich/HumanPractice/Interviews2013-10-29T03:38:48Z<p>FlorianA: </p>
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==Expert Counsel: An overview==<br />
All throughout the development process of our PhyscoFilter we got in touch with experienced experts and stakeholders to constantly improve our technical solution and to assess potential users demands on its properties.<br />
<br />
[[File:TUM13_Spread_the_word.png|thumb|center|910px|'''Figure 1:''' Overview of our interviews]]<br />
<br />
==1) 4<sup>th</sup> Water Research Horizon Conference “Water Scarcity and Global change” in Berlin==<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference.JPG|thumb|right|350px|'''Figure 2''': 4th Water Research Horizon Conference “Water Scarcity and Global change” in Berlin]]<br />
The main goal of the participation on the 4th [http://www.ufz.de/water-research-horizon Water Research Horizon Conference] “Water Scarcity and Global change” in Berlin on the 25th of June 2013 was to inform ourselves about worldwide water scarcity and global change. We hoped to get inspirations for our '''outreach''' and '''human practice''' effort. Moreover we were interested in the worldwide practical realization of minimizing “the water problem”. Especially the diverse ways of approach in different countries and the existing solutions were of interest (see Figure 2) to see how our project fits into this scope. In addition to the main speeches of ambassadors from different communities and companies we participated on a workshop session with speeches and discussion called ''“Water and Soils for Food and Energy - A Question of '''Technological Development'''?”''. <br />
<br />
We realized that it is quite difficult to give a straight answer to this question. J. Bouma (formerly Wageningen University, The Netherlands) answered with ''“No, not alone, but start with existing technology and go from there”''. Whereas R. Ardakanian (United Nations University, Dresden) just said ''“yes”'' after his speech. But not only the answers on the topic question differed, also the point of view concerning to the worldwide water problem, scarcity and the definition of common water diverged. In his speech, R. Ardakanian placed '''water''' in context with '''waste'''. He said that waste can be a resource for the production of food and energy and must contribute as '''recycled''' waste in technology. According to E. Adar the goal and challenge for the next generation is (Ben Gurion University, Negev, Israel) minimizing the water use and costs. To him it was evident that water is a synonym for food and a commodity, not a resource like gold.<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference_speech_1.JPG|thumb|left|350px|'''Figure 3''': speech about ''"historic and future variations in global water"'' from J. Sheffield]]<br />
<br />
Furthermore M. Lange (The Cyprus Institute, Nicosia) presented his speech titled ''"Ways about Climate Change and Energy-Water Nexus in the Mediterranean"''. A. Porporato (Duke University, NC, USA) gave a lecture on the role of '''ecohydrological variability''' in productivity and sustainability of ecosystems.<br />
<br />
The following general speeches about the water scarcity and climate change, informed the audience about different present projects to reduce the water scarcity. G. Teutsch (UFZ Leipzig) who opened the conference talked about a ''“quick start”'' to reduce the water scarcity, which sticks together with the urgent topic of '''climate change'''. The main statement of the second speaker's speech ,P. Krebs (TU Dresden), was that not visions yield viable results, but only excellent '''concepts''', new '''projects''' and '''community action'''. That the water topic has also an important role in politics, was approached from F. Holzwarth, (BMU Bonn) whose goal is forming a ''“water alliance with new individual faces”''. He stated that in contrast to the climate change, the water scarcity isn’t a major topic in politics, because ''“disasters drive '''politics'''”''. In his speech U. Weber (DFG Bonn) picked out science (project, implementation and ideas) and society (public, politic) in relation to water scarcity and climate change as central themes. In turn, J. Sheffield (Princeton University, USA; see Figure 3) broached the issues of the transition between drought and floods.<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference_speech_2.JPG|thumb|right|350px|'''Figure 4''': speech about addressing our emerging water futures:The water-agriculture-energy-ecosystem nexus from G.Bidoglio]]<br />
The theoretical aspect of controlling natural water's quality in a '''mathematical model''' was presented by G. Bidoglio(EC-JRC IES Ispra, Italy; see figure 4). Implemented Projects are the Joint Research Project SMART (integrated Water Resource Management in the Lower Jordan Valley) and the Guanting Project (sustainable water and agricultural land use in the Guanting watershed under limited water resources).<br />
<br />
In summary the visit of the conference made us realize that water '''scarcity''' and '''water quality''' are in fact vital problems that already attract much attention in academic circles. In contrast, we did not know that the global water situation is such a multifaceted subject and we were stunned by the broad range of subjects that were covered during the conference. As the conference took place at the beginning of our project, the impressions we received from the conference helped us '''understanding''' what problems we should address when talking to a broader audience in our subsequent human practice efforts.<br />
Moreover the presentation of currently happening innovative projects '''inspired''' us in the design of our filter prototypes.<br />
<br />
==2) Workshop "Drugs in the Environment" in Berlin organized by the RCDS==<br />
[[File:TUM13_RCDS-conference.png|thumb|right|200px|'''Figure 5''': Description]]<br />
At the end of May we joined the "Drugs in the environment" conference that was organized by the [http://www.rcds.de/ RCDS] (which is the youth organisation of the CDU, the currently governing political party in Germany) and the [http://www.vfa.de/ German Association of Research-Based Pharmaceutical Companies].<br> <br />
During this discussion meeting we had the opportunity to meet and discuss with several experts as well as with representatives of leading German pharmacy companies such as Bayer and Novartis. The main focus of this discussion was on the point whether '''pollution''' of the environment by '''pharmaceutic substances''' is a considerable problem for our society. Furthermore whether Germany is going to '''invest money''' to reduce this pollution and who is responsible for financing these measures. The conclusion of this conference was that in Germany there is no acute danger for mankind by contamination of the environment by pharmaceutic substances. In contrast the lowering of the detection limit for several substances leads to an '''awareness''' that there is contamination caused by mankind and that several people are not willing to accept this pollution.<br />
<br />
==3) Biotechnology 2020+ Conference of the Ministry of Research and Education in Berlin==<br />
<br />
[[File:TUM13_Biotechnology 2020+.jpg|thumb|right|350px|'''Figure 6''': Biotechnology 2020+ Conference of the Ministry of Research and Education: first discussion round about the topic “result of the previous activities and recommendations for action for the continuation of the initiative "Biotechnology 2020+ conference" with the five ambassadors of '''five research institutions''' ( Frauenhofer Society, Helmholtz Association, Max-Planck Society, Leibniz Association, TU Braunschweig)]]<br />
For the third year in a row the German Ministry of Research and Education invited all German iGEM Teams to join the "[http://www.biotechnologie2020plus.de Biotechnology 2020+ conference]" in Berlin on June 27th. ''“Which products and biotechnological processes are fundamental? “'', ''“How will the next generation accept the biotechnological processes?”'' and ''“Which projects are already initiated?”''. Experts in the fields economy, politics and science and the German iGEM teams came together at the conference and tried to answer these questions. In his speech, Dr. Helge Braun (parliamentary State Secretary of the BMBF) emphasized that a creative teamwork with an own developed ''"language"'' is necessary. He introduced the new biotechnological ideas: biological implants, biomimetic desalination plants and microbial fuel cells. In the first discussion round about the topic “result of the '''previous activities''' and '''action recommendations''' for the continuation of the initiative "Biotechnology 2020+ conference", five ambassadors of five research institutions (Frauenhofer Society, Helmholtz Association, Max-Planck Society, Leibniz Association, TU Braunschweig) (see figure 5) presented their '''recently''' implemented '''ideas''': printable biotechnology, cell-free bioproduction and synthesis of novel therapeutics. Exemplarily Prof. Dr. H. Jäckle (Max-Planck-Community) introduced the research network for Synthetic Biology MaxSynbio. The second discussion treated the subject "Open Source in the Biotechnology”. After the presentation there were workshops on the topic “On the way to a Biotechnology 2020+”. Additionally the German iGEM projects 2013 were exhibitied (see Figure 6) and the five research institutions presented their recent research projects.<br />
[[File:TUM13_Biotechnology 2020+ iGEM German teams.jpg|thumb|left|350px|'''Figure 7''': Biotechnology 2020+ Conference of the Ministry of Research and Education: exhibition of the German IGEM projects 2013]]<br />
<br />
In summary the participation informed us about the practicable operated '''novel biotechnology ideas'''. It enabled our cooperations and gave us an '''insight''' into projects from market-leading research institutions in Germany. Furthermore we got in contact with other iGEM teams which paved the way for an iteration of the German iGEM day. <br />
<br />
[[File:TUM13_Biotechnology 2020_Poster.png|thumb|right|300px| '''Figure 8''': Poster presentation of our PhyscoFilter. ]]<br />
<div class="visualClear"></div><br />
<br />
==4) Visit of the Helmholtz Center for Environmental Research (UFZ) in Leipzig ==<br />
<br />
[[File:TUM13_Visit of the UFZ.JPG|thumb|right|350px|'''Figure 9''': Visit of the UFZ: Johanna and Andreas from iGEM TU Munich team with Dr. Kuschk (left) and Dr. Müller (right)]]<br />
When we visited the Helmholtz Zentrum of Leipzig [http://www.ufz.de/index.php?en=2540 UFZ] on the 26 th of June 2013, we had three objectives. We wanted to '''present our project''', get another impartial '''opinion''' concerning our project idea and project implementation and lastly acquire '''general information''' about practical phytoremediation. Dr. Kuschk and Dr. Müller are chair members of the environmental biotechnology section and attend to '''phytoremediation''' or rather bioremediation (see figure 6). Their research focus lies in the area of zoonosis that means the interplay between plants and microorganisms. The plant acts with the root system as a carrier to quantify the microorganisms which dispose nitrate, sulfur or toluol. According to Dr. Kuschk genetic engineering work isn’t possible at the UFZ, because of the few personnel at the moment.<br />
<br />
<br />
Dr. Müller gave us a guided tour through the laboratories. (see Figure 7) Later on we had two conversations with experts on different issues concerning our project. Dr. Müller gave us very helpful annotations about our '''effectors'''. We were explained the sense of '''amplifying the resistance gene EreB''' in environment in relation to the biotechnological benefit. He informed us about the costs of blue light. Furthermore he alluded that we had to consider the '''enzyme specification''' in order to find the benefit of extincting erythromycin to clean water. Another point was the use of '''cathecholdioxygenase'''. According to Dr. Müller, depletion of catechol through microorganisms is a very effective process. So our next step was to show the efficiency of the depleting catechol through moss. <br />
<br />
[[File:TUM13_Visit of the UFZ laboratories.JPG|thumb|left|350px|'''Figure 10''': Visit of the UFZ: Dr. Müller leads two iGEM TU Munich team members through the laboratories.]]<br />
<br />
The main topic we discussed with Dr. Kuschk was the application of our project idea in '''practice''', outside the competition framework. Dr. Kuschk was in '''doubt''' about the practical implementation. His assessment was based on his comprehensive experiences with concepts and conversions he developed. In his opinion the '''costs''', the low concentration of polluting substances and the small contact surface have negative effects on the projects application. When it comes to the immobilization of substances, he prefers biofilms to membrane bounds. He also criticized our choice of degraded substances. In his opinion fecal substances are the cause of the high death and diseases rate in less developed countries. We explained that we chose harmful substances, according to the WHO, to establish our filter, as we'd like to show the '''degradation and bioaccumulation''' of them. In general he considered our project as basic research and not as practical application.<br />
In summary the visit of the UFZ enriched our knowledge in the issue of molecular genetics and gave us an idea how further project thoughts could look like. The guided tour through the laboratories of UFZ showed us '''execution examples''' of working phytoremediation concepts.<br />
<br />
==5) Expert Box: [http://www.pieris-ag.com Pieris AG]==<br />
[[File:TUM13_Expert_pieris.png|thumb|left|350px| '''Figure 11''': Expert interview with the [http://www.pieris-ag.com Pieris AG]]]<br />
The [http://www.pieris-ag.com Pieris AG] is a '''biopharmaceutical company''' founded in 2001 which is commercializing the '''anticalin''' production technology. Anticalins are designed to '''bind proteins''' that are a '''promising alternatives to antibodies'''. These are mainly used in red biotechnology. They are, for example, used in tumor therapy. As these engineered binding proteins can be directed against nearly every chemical structure, they are also an interesting tool for phytoremediation. Our idea was to apply the successful anticalin technology in a totally different context, namely for the '''detoxification of aquatic ecosystems'''. We contacted the Principal Scientist of the [http://www.pieris-ag.com Pieris AG] and they are confident that a meeting will be possible until the regional Jamboree in Lyon.<br />
<br />
==6) Expert Box: Prof. Reski (Freiburg University)==<br />
[[File:TUM13_expert_Reski.png|thumb|450px|left| '''Figure 12:''' Expert interview with Prof. Reski. ]]<br />
When we had chosen phytoremediation as our topic for this year's competition it became clear soon that ''Physcomitrella patens'' is a '''great chassis''' which could bring iGEM closer to the real world applications we are looking for. For this reason we contacted Prof. Dr. Reski who is a worldwide expert on ''Physcomitrella patens'' and especially its biotechnological application. He was very kind and liked our idea from the first moment on. Especially when we told him to introduce '''his ''Physco'' '''to iGEM. So he offered us help where ever we would need it. In the end we traveled five times to Freiburg (350 km) with different team members to perform the transformations in his lab. '''Fetching plasmids''' which are known to be functional in ''Physcomitrealla patens'' or just to discuss with him or his co-workers. We could win him as an '''advisor''' for our team. That is especially pleasant as iGEM starts bringing together universities and scientific groups from different places and and our iGEM team was not restricted to our own university.<br />
<br />
==7) Visiting the iGEM Team Freiburg 2013==<br />
[[File:Muenchen-Freiburg-2013.jpg|thumb|left|350px|'''Figure 5:''' iGEM Teams Freiburg and Munich joint during our first visit]]<br />
[[File:TUM13_Freiburg.JPG|thumb|right|200px|'''Figure 6:''' Our second visit]]<br />
During our transformation experiments at Prof. Reski's lab we spent more than one evening with the iGEM team Freiburg and had for example a very delicious barbecue and an interesting scientific exchange. Thank you a lot for your hospitality, congratulations for your success in Lyon and see you in '''Boston'''!<br />
<br />
==8) Expert Box: Greenovation (Freiburg)==<br />
<br />
[[File:TUM13_expert_Greenovation.png|thumb|450px|left| '''Figure 13:''' Expert interview with Dr. Schaaf, Principal Scientist at [http://www.greenovation.com/index.html Greenovation BioTech GmbH]]]<br />
In September we went on a road trip to visit interesting companies and experts to get some consultation on our plans. As there is a biotech company which successfully uses ''Physcomitrella patens'' as a '''production organism''' for '''therapeutic proteins''' it became clear that we had to meet a responsible person of this company. Dr. Schaaf, the Principal Scientist of [http://www.greenovation.com/index.html Greenovation] invited us for a discussion and subsequently he showed us the production facility and the other labs of Greenovation. During the meeting we discussed their work flow for ''Physcomitrella'' and ours. We talked about '''production rates''' of therapeutic proteins and learned about its '''cultivation conditions''' in a bioreactor as well as in nature. During the discussion we also considered ordering the transformation of PhyscoFilter plants (so calles BryoTechnology). This '''hypothetical collaboration''' would have been possible and would have cost us around 50,000 to 70,000€ per effector. The price would have included the '''DNA preparation''', '''transformation''' and '''expression analysis''' of 1000 clones. Beside the possibility of a collaboration with PhyscoFilter the company also holds '''patents''' which could, in theory, block the application of ''Physcomitrella'' for commercial phytoremediation. In this case it would become necessary to buy a license for the usage of transgenic ''phycomitrella'' plants. Other experts we have consulted can be found in our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews Advisory Board].<br />
<br />
==9) Expert Box: Prof. Posten (KIT Karlsruhe)==<br />
During our road trip we talked to Prof. Dr.-Ing. Clemens Posten, who is head of the [http://bvt.blt.kit.edu/ Institute of bioprocess engineering] at the Karlsruhe Institute of Technology (KIT). During our visit we were shown the institutes's bioreactors and Prof. Posten gave us an idea of how a '''symbiosis between plant and technology''' can look like. In the past his group worked on a collaboration project with Prof. Dr. Reski (see our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews Advisery Board]) on biological process engineering for ''Physcomitrella patens''. As part of this discussion we figured out several important parameters to control and possible problems to solve in order to successfully implement our ''PhyscoFilter''. <br><br />
Read more about the results of this [https://2013.igem.org/Team:TU-Munich/Project/Implementation consultation].<br />
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==10) Visiting the iGEM Team Heidelberg 2013==<br />
On our road trip we also spent an evening with the iGEM team '''[https://2013.igem.org/Team:Heidelberg Heidelberg 2013]'''. At first they told us that they were too busy with experiments but in the end they found 3 team members who showed us their lab and the gorgeous city of Heidelberg. Thank you a lot and see you in '''Boston'''!<br />
<br />
==11) Expert box: Bestmann Green Systems (near Hamburg)==<br />
[[File:TUM13_expert_Bestemann.png|thumb|350px|left| '''Figure 14:''' ]]<br />
[http://www.bestmann-green-systems.de/ Bestmann Green Systems] is an engineering company which offers commercial solutions for '''embedding vegetation in aquatic ecosystems'''. As this is a major point of our implementation project we also talked to this company (because of the actual distance a visit was not possible). This company also sells '''swimming raft''' devices which allow the cultivation of plants on free water. This technology was highly interesting also for our [https://2013.igem.org/Team:TU-Munich/Results/Economics business model] in which [http://www.bestmann-green-systems.de/ Bestmann Green Systems] could be a potential collaboration partner.<br />
<br />
==12) Discussion of our Project with Dr. Christoph Then from Testbiotech==<br />
Dr. Christoph Then is a recognized expert on '''risk assesment''' in the biotechnological field and the CEO of the [http://www.testbiotech.org/ Testbiotech registered association]. Amongst many other engagements, he has worked as an expert advisor on agriculture and genetic engineering for the '''Green Party''' in the Bavarian Landtag (legislative assembly in a German state) and as expert for '''Greenpeace on agriculture, genetic engineering and consumer affairs'''. Since our iGEM project deals with Green Biotechnology and since our '''various implementation ideas include biosafety and GMO release aspects''', he was the ideal expert to talk to. We had a very productive and interesting discussion that helped us get a much clearer picture on these aspects as well as on the '''regulations of GMO authorisation'''.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/HumanPractice/OverviewTeam:TU-Munich/HumanPractice/Overview2013-10-29T03:37:28Z<p>FlorianA: </p>
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== Human Practice Overview ==<br />
We know about the great importance of communication in science and the big role it plays in the fate of new technologies. Therefore we set out to collect lots of valuable feedback, to share our ideas and to inspire new perspectives. On this page you can read about our efforts to interact with experts and get in touch with the public. We learned a lot from the experts and they encouraged us to further improve our project. We also hope that we arouse the public interest in synthetic biology as its importance is likely to rise over the next years.<br />
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===Expert Counsel===<br />
We traveled Germany seeking advice from professors, talking to experts from biotechnologic companies, meeting with professionals at several conferences to talk about our project and getting in touch with other iGEM teams.<br>[https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews Read more]<br />
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===GMO Release===<br />
We analyzed the risks and benefits of our project and collected background information about the current legal situation and ethical aspects.<br>[https://2013.igem.org/Team:TU-Munich/HumanPractice/GMO Read more]<br />
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===Publicity===<br />
We presented our project to the public, got into the public discussion on genetic engineering in Germany and were covered in the media.<br>[https://2013.igem.org/Team:TU-Munich/HumanPractice/Media Read more]<br />
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===Educational Kit===<br />
We also created educational kits for teachers to conduct a basic biotechnological experiment with their students.<br>[https://2013.igem.org/Team:TU-Munich/HumanPractice/Education Read more]<br />
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===Tutorials===<br />
We would like to share the experiences we made and pass on the solutions and skills we generated this summer with the iGEM community, so we created a couple of useful tutorials for the following iGEM generations.<br><br />
[https://2013.igem.org/Team:TU-Munich/Results/How_To Read more]<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Modeling/Protein_PredictionsTeam:TU-Munich/Modeling/Protein Predictions2013-10-29T03:33:47Z<p>FlorianA: /* References: */</p>
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==Prediction of Protein Structures and Functions==<br />
'''Structural properties''' of '''effector proteins''' are often essential for their function, so it is advantageous to know about them. It is for example necessary to know whether '''termini are accessible''' for protein fusion or whether the protein is '''only functional in a multimeric fold'''. For this reason a structure based search was performed in the [http://www.rcsb.org/pdb/home/home.do protein database]. As the number of identified structures is still limited, it is a promising attempt to look for homologous proteins whose crystal structures have been determined.<br />
<br />
==Analysis of Receptor Sequences &ndash; Choosing the right template ==<br />
<br />
For several purposes of our project, we needed a synthetic receptor enabling us to integrate proteins into the membrane in the desired orientation, i.e. to express protein-domains on the intracellular or extracellular side of the cell membrane. We investigated several different plant-receptors from the well characterized dicotyledon ''Arabidopsis thaliana'' and the moss ''Physcomitrella patens'', our chassis. The receptors from ''Arabidopsis thaliana'' have the advantage that their transgenic expression has successfully been demonstrated [[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] whereas the native receptors from ''Physcomitrella patens'' bear only a small risk of failing [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]].<br><br />
Due to the fact that there were many different available receptors, which we could have used as template for our synthetic receptor, we used bioinformatical methods to evaluate the suitability of these receptors. The following three examples ERF, FLS2 and SERK shown in table 1 resulted from this equation.<br />
<br />
{|cellspacing="0" border="1"<br />
|+ '''Table 1:''' Examined Receptors<br />
!Receptor<br />
!Organism<br />
!Length (aa)<br />
!Sequence reference<br />
!Literature reference<br />
|-<br />
|ERF<br />
|''A. thaliana''<br />
|1031<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_197548.1 NP_197548.1]<br />
|<br />
|-<br />
|FLS2<br />
|''A. thaliana''<br />
|1173<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_199445.1 NP_199445.1]<br />
|<br />
|-<br />
|SERK<br />
|''P. patens''<br />
|625<br />
|[http://www.ncbi.nlm.nih.gov/protein/XP_001759122.1 XP_001759122.1]<br />
|[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]<br />
|-<br />
|}<br />
<br />
===Prediction of Signal Peptides===<br />
[[File:TUM13 Modeling_Signal-P.png|thumb|right|350px| '''Figure 1:''' Prediction and analysis of signal peptides]]<br />
'''Introduction'''<br><br />
A first analysis was performed to identify signal-peptides, which are bound by the cellular signal recognition particle and lead to the translocation of the bound polypeptide into the endoplasmic reticulum. Afterwards the signal peptide is cleaved by a signal peptide peptidase at a specific site. The analysis for signal peptides was done by using the [http://www.cbs.dtu.dk/services/SignalP SignalP 4.1 Server]. <br><br />
<br><br />
'''Results'''<br><br />
The program was run for different receptors and will be illustrated for the three examples mentioned above (see fig. 1). <br><br />
The figure shows the N-terminal sequence of the receptors, together with three scores: <br><br />
(1) The C-Score (raw cleavage site score) in red. <br><br />
(2) The S-Score (signal peptide score) in green. <br><br />
(3) The Y-Score (combined cleavage site score) in blue.<br><br />
<br><br />
The C-Score shows the most probable cleavage site identified by the peptidase. It was possible to identify the most probable cleavage site for all shown receptors with ambiguous cleavage sites for the SERK-receptor. The amino acid with the highest C-score is predicted to be the first amino acid of the primary structure of the cleaved receptor. <br><br />
The S-Score was developed to identify amino acid sequences which appear in a signal peptide and others that belong to the matured receptor. The course of this parameter is high for the first 23-28 amino acids of all receptors, identifying these residues as signal peptides. The amino acid residue, which lies at the greatest decrease of the S-Score, is the predicted border between the N-terminal signal peptide and the receptor. <br><br />
The Y-Score results from the geometrical structure of the protein and the previously determined scoring parameters. It illustrates that the two first parameters show a good fit for the identification of the signal peptide in all three indicated receptors.<br><br />
<br><br />
'''Discussion'''<br><br />
Summarizing these parameters, it can be concluded that all three pictured receptors seem to contain a sequence acting as a signal peptide. For many of the predicted receptors in the genome of ''Physcomitrella patens'' the prediction did not yield a positive result. With respect to the signal peptide, all mentioned receptors would be suitable as a template for our synthetic receptor. The predicted data show that the SERK-Receptor is favorable for our application, because its signal peptide is statistically the best recognized one and bears the smallest risk of failure.<br />
<br />
<br><br />
<br><br />
<br />
===Prediction of Transmembrane Regions===<br />
[[File:TUM13 Modeling_TMHMM.png|thumb|right|350px| '''Figure 2:''' Prediction and analysis of transmembrane regions]]<br />
'''Introduction'''<br><br />
Additional to the identification of the signal peptide, it was very important to identify transmembrane regions within the receptors, because we wanted to use a type I receptor as a template that contains a N-terminal extracellular domain, a transmembrane domain and a C-terminal intracellular domain (see [https://2013.igem.org/Team:TU-Munich/Project/Localisation our localization page]). To analyze this issue, the prediction tool [http://www.cbs.dtu.dk/services/TMHMM TMHMM] was used for several different receptors. Again the most suitable receptors were ERK, FLS2 and SERK.<br><br />
<br><br />
'''Results'''<br><br />
The analysis yields a signal peptide and a single transmembrane domain for all three depicted receptors (see fig. 2). The estimated reliability of the predictions was equally good for all examined receptors, whereas the signal peptide was most reliably predicted for the SERK receptor. <br><br />
<br><br />
'''Discussion'''<br><br />
Focussing on the membrane topology point of view, all the investigated receptors would be suitable blue prints for our synthetic receptor. As the SERK-Receptor yields the best prediction, it was chosen as the favorable template. Another reason to choose the SERK-Receptor was that it is derived from ''Physcomitrella patens''. The only problem, concerning this prediction, is that the N-terminus of this receptor is predicted to be extracellular. The falsification of this prediction was simple, because the SERK receptor contains a C-terminal kinase-domain, which is known to be involved in signal transduction.<br />
<br><br />
<br><br />
<br />
===Choice of the SERK Receptor===<br />
Finally we decided to use the SERK receptor as a template to generate our synthetic receptor. The final receptor was designed in RFC[25] standard, which allows in frame protein fusions. The final constructs were designed containing the SERK signal peptide ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]), an extracellularly located effector protein, the transmembrane domain of the SERK receptor ([http://parts.igem.org/Part:BBa_K1159305 BBa_K1159305]), a short linker and a GFP, to investigate the cellular localization of our receptor with the aid of fluorescence microscopy.<br />
<br />
==Searching for Homologous Structures using HHpred==<br />
<br />
The search for homologous structures was performed by using the freely accessible web server [http://toolkit.tuebingen.mpg.de/hhpred HHpred] [[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]]. The amino acid sequences for the BioBricks were translated into amino acid sequences using the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] and was then inserted into the the search field. The results for all proteins investigated in our project are shown in table 2. <br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 2:''' Predicted Structures<br />
!Protein<br />
!BioBrick<br />
!PDB-code<br />
!Identity<br />
!Similarity<br />
!Structure<br />
|-<br />
|XylE<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K147002 BBa_K147002]<br />
| [http://www.rcsb.org/pdb/explore.do?structureId=3hpy 3hpy_A]<br />
|50%<br />
|0.939<br />
|[[File:TUM13 small_XylE.png|85px]]<br />
|-<br />
|Laccase<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159002 BBa_K1159002]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2wsd 2wsd_A]<br />
|68%<br />
|1.223<br />
|[[File:TUM13 small_Laccase.png|85px]]<br />
|-<br />
|NanoLuc<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159001 BBa_K1159001]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3ppt 3ppt_A]<br />
|21%<br />
|0.359<br />
|[[File:TUM13 small_NanoLuc.png|85px]]<br />
|-<br />
|EreB<br />
|[http://parts.igem.org/Part:BBa_K1159000 BBa_K1159000]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3b55 3b55_A]<br />
|19%<br />
|0.318<br />
|[[File:TUM13_small_EreBx.png|85px]]<br />
|-<br />
|Spycatcher<br />
|[http://parts.igem.org/Part:BBa_K1159200 BBa_K1159200]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2x5p 2x5p_A]<br />
|97%<br />
|1.298<br />
|[[File:TUM13 small_SpyCatcher.png|85px]]<br />
|-<br />
|PP1<br />
|[http://parts.igem.org/Part:BBa_K1159004 Part:BBa_K1159004]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3e7a 3e7a_A]<br />
|96%<br />
|1.593<br />
|[[File:TUM13_small_PP1.png|85px]]<br />
|-<br />
|GFP<br />
|[http://parts.igem.org/Part:BBa_K1159311 BBa_K1159311]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2WUR 2WUR]<br />
|98%<br />
|1.477<br />
|[[File:TUM13 small_GFP.png|85px]]<br />
|-<br />
|Glutathiontransferase / DDT Dehydrochlorinase<br />
|<partinfo>BBa_K620000</partinfo><br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3F6D 3F6D]<br />
|68%<br />
|1.155<br />
|[[File:TUM13 small_GST.png|85px]]<br />
|-<br />
|SERK-TM<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159305 BBa_K1159305]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2ks1 2ks1_B]<br />
|24%<br />
|0.233<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|TEV Protease<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=1Q31 1Q31]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|Streptavidin<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3RY2 3RY2]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|}<br />
<br />
====Results====<br />
The homology search showed that some of our effector proteins have very closely related proteins with a known structure. For example there are very similar protein structures available for the SypCatcher, PP1 and GFP each with identities of over 90%. Some other effector proteins such as XylE, Laccase or the DDT Dehydrochlorinase have less homologous proteins, whose structures still give good hints on structural questions. However there are also effectors where only badly matched structures are known, which can only be used as a very rough indication of the fold. The NanoLuc luciferase, which is a highly engineered protein derived from shrimps and was only published this year, is an example of a protein with no known structural homologue.<br><br />
The structures obtained here were used to design our experiments. A homology modeling for the Laccase was performed to determine whether it contains disulphide bridges. The resulting homologous structures were used as illustrations, as explained in one of our [https://2013.igem.org/Team:TU-Munich/Results/How_To How-Tos] about animated GIFs.<br />
<br />
==References:==<br />
[[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] MARGARET T. Boylan, M.T. and Quail, P.H. (1991). PhytochromeA overexpression inhibits hypocotyl elongation in transgenic ''Arabidopsis''. ''Proc. Natl. Acad. Sci.'' 88:10806-10810.<br><br />
<br />
[[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] Reski, R. (1998). Development, Genetics and Molecular Biology of Mosses. ''Bot. Acta'', 111:1-15.<br /><br />
<br />
[[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]] Lienhart, O. (2007). Untersuchungen zu einem Somatic-Embryogenesis-Receptor-like-Kinase-Homolog in ''Physcomitrella patens'' (Hedw.) B.S.G. PhD-thesis at Freiburg University<br />
<br />
[http://www.cbs.dtu.dk/services/SignalP/ SignalP41] Server<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]] Söding J, Biegert A, Lupas AN. (2005). The HHpred interactive server for protein homology detection and structure prediction. ''Nucleic Acids Res.'' 2005 Jul 1;33(Web Server issue):W244-8.<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Modeling/Protein_PredictionsTeam:TU-Munich/Modeling/Protein Predictions2013-10-29T03:33:24Z<p>FlorianA: /* Analysis of Receptor Sequences &ndash; Choosing the right template */</p>
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==Prediction of Protein Structures and Functions==<br />
'''Structural properties''' of '''effector proteins''' are often essential for their function, so it is advantageous to know about them. It is for example necessary to know whether '''termini are accessible''' for protein fusion or whether the protein is '''only functional in a multimeric fold'''. For this reason a structure based search was performed in the [http://www.rcsb.org/pdb/home/home.do protein database]. As the number of identified structures is still limited, it is a promising attempt to look for homologous proteins whose crystal structures have been determined.<br />
<br />
==Analysis of Receptor Sequences &ndash; Choosing the right template ==<br />
<br />
For several purposes of our project, we needed a synthetic receptor enabling us to integrate proteins into the membrane in the desired orientation, i.e. to express protein-domains on the intracellular or extracellular side of the cell membrane. We investigated several different plant-receptors from the well characterized dicotyledon ''Arabidopsis thaliana'' and the moss ''Physcomitrella patens'', our chassis. The receptors from ''Arabidopsis thaliana'' have the advantage that their transgenic expression has successfully been demonstrated [[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] whereas the native receptors from ''Physcomitrella patens'' bear only a small risk of failing [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]].<br><br />
Due to the fact that there were many different available receptors, which we could have used as template for our synthetic receptor, we used bioinformatical methods to evaluate the suitability of these receptors. The following three examples ERF, FLS2 and SERK shown in table 1 resulted from this equation.<br />
<br />
{|cellspacing="0" border="1"<br />
|+ '''Table 1:''' Examined Receptors<br />
!Receptor<br />
!Organism<br />
!Length (aa)<br />
!Sequence reference<br />
!Literature reference<br />
|-<br />
|ERF<br />
|''A. thaliana''<br />
|1031<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_197548.1 NP_197548.1]<br />
|<br />
|-<br />
|FLS2<br />
|''A. thaliana''<br />
|1173<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_199445.1 NP_199445.1]<br />
|<br />
|-<br />
|SERK<br />
|''P. patens''<br />
|625<br />
|[http://www.ncbi.nlm.nih.gov/protein/XP_001759122.1 XP_001759122.1]<br />
|[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]<br />
|-<br />
|}<br />
<br />
===Prediction of Signal Peptides===<br />
[[File:TUM13 Modeling_Signal-P.png|thumb|right|350px| '''Figure 1:''' Prediction and analysis of signal peptides]]<br />
'''Introduction'''<br><br />
A first analysis was performed to identify signal-peptides, which are bound by the cellular signal recognition particle and lead to the translocation of the bound polypeptide into the endoplasmic reticulum. Afterwards the signal peptide is cleaved by a signal peptide peptidase at a specific site. The analysis for signal peptides was done by using the [http://www.cbs.dtu.dk/services/SignalP SignalP 4.1 Server]. <br><br />
<br><br />
'''Results'''<br><br />
The program was run for different receptors and will be illustrated for the three examples mentioned above (see fig. 1). <br><br />
The figure shows the N-terminal sequence of the receptors, together with three scores: <br><br />
(1) The C-Score (raw cleavage site score) in red. <br><br />
(2) The S-Score (signal peptide score) in green. <br><br />
(3) The Y-Score (combined cleavage site score) in blue.<br><br />
<br><br />
The C-Score shows the most probable cleavage site identified by the peptidase. It was possible to identify the most probable cleavage site for all shown receptors with ambiguous cleavage sites for the SERK-receptor. The amino acid with the highest C-score is predicted to be the first amino acid of the primary structure of the cleaved receptor. <br><br />
The S-Score was developed to identify amino acid sequences which appear in a signal peptide and others that belong to the matured receptor. The course of this parameter is high for the first 23-28 amino acids of all receptors, identifying these residues as signal peptides. The amino acid residue, which lies at the greatest decrease of the S-Score, is the predicted border between the N-terminal signal peptide and the receptor. <br><br />
The Y-Score results from the geometrical structure of the protein and the previously determined scoring parameters. It illustrates that the two first parameters show a good fit for the identification of the signal peptide in all three indicated receptors.<br><br />
<br><br />
'''Discussion'''<br><br />
Summarizing these parameters, it can be concluded that all three pictured receptors seem to contain a sequence acting as a signal peptide. For many of the predicted receptors in the genome of ''Physcomitrella patens'' the prediction did not yield a positive result. With respect to the signal peptide, all mentioned receptors would be suitable as a template for our synthetic receptor. The predicted data show that the SERK-Receptor is favorable for our application, because its signal peptide is statistically the best recognized one and bears the smallest risk of failure.<br />
<br />
<br><br />
<br><br />
<br />
===Prediction of Transmembrane Regions===<br />
[[File:TUM13 Modeling_TMHMM.png|thumb|right|350px| '''Figure 2:''' Prediction and analysis of transmembrane regions]]<br />
'''Introduction'''<br><br />
Additional to the identification of the signal peptide, it was very important to identify transmembrane regions within the receptors, because we wanted to use a type I receptor as a template that contains a N-terminal extracellular domain, a transmembrane domain and a C-terminal intracellular domain (see [https://2013.igem.org/Team:TU-Munich/Project/Localisation our localization page]). To analyze this issue, the prediction tool [http://www.cbs.dtu.dk/services/TMHMM TMHMM] was used for several different receptors. Again the most suitable receptors were ERK, FLS2 and SERK.<br><br />
<br><br />
'''Results'''<br><br />
The analysis yields a signal peptide and a single transmembrane domain for all three depicted receptors (see fig. 2). The estimated reliability of the predictions was equally good for all examined receptors, whereas the signal peptide was most reliably predicted for the SERK receptor. <br><br />
<br><br />
'''Discussion'''<br><br />
Focussing on the membrane topology point of view, all the investigated receptors would be suitable blue prints for our synthetic receptor. As the SERK-Receptor yields the best prediction, it was chosen as the favorable template. Another reason to choose the SERK-Receptor was that it is derived from ''Physcomitrella patens''. The only problem, concerning this prediction, is that the N-terminus of this receptor is predicted to be extracellular. The falsification of this prediction was simple, because the SERK receptor contains a C-terminal kinase-domain, which is known to be involved in signal transduction.<br />
<br><br />
<br><br />
<br />
===Choice of the SERK Receptor===<br />
Finally we decided to use the SERK receptor as a template to generate our synthetic receptor. The final receptor was designed in RFC[25] standard, which allows in frame protein fusions. The final constructs were designed containing the SERK signal peptide ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]), an extracellularly located effector protein, the transmembrane domain of the SERK receptor ([http://parts.igem.org/Part:BBa_K1159305 BBa_K1159305]), a short linker and a GFP, to investigate the cellular localization of our receptor with the aid of fluorescence microscopy.<br />
<br />
==Searching for Homologous Structures using HHpred==<br />
<br />
The search for homologous structures was performed by using the freely accessible web server [http://toolkit.tuebingen.mpg.de/hhpred HHpred] [[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]]. The amino acid sequences for the BioBricks were translated into amino acid sequences using the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] and was then inserted into the the search field. The results for all proteins investigated in our project are shown in table 2. <br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 2:''' Predicted Structures<br />
!Protein<br />
!BioBrick<br />
!PDB-code<br />
!Identity<br />
!Similarity<br />
!Structure<br />
|-<br />
|XylE<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K147002 BBa_K147002]<br />
| [http://www.rcsb.org/pdb/explore.do?structureId=3hpy 3hpy_A]<br />
|50%<br />
|0.939<br />
|[[File:TUM13 small_XylE.png|85px]]<br />
|-<br />
|Laccase<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159002 BBa_K1159002]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2wsd 2wsd_A]<br />
|68%<br />
|1.223<br />
|[[File:TUM13 small_Laccase.png|85px]]<br />
|-<br />
|NanoLuc<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159001 BBa_K1159001]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3ppt 3ppt_A]<br />
|21%<br />
|0.359<br />
|[[File:TUM13 small_NanoLuc.png|85px]]<br />
|-<br />
|EreB<br />
|[http://parts.igem.org/Part:BBa_K1159000 BBa_K1159000]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3b55 3b55_A]<br />
|19%<br />
|0.318<br />
|[[File:TUM13_small_EreBx.png|85px]]<br />
|-<br />
|Spycatcher<br />
|[http://parts.igem.org/Part:BBa_K1159200 BBa_K1159200]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2x5p 2x5p_A]<br />
|97%<br />
|1.298<br />
|[[File:TUM13 small_SpyCatcher.png|85px]]<br />
|-<br />
|PP1<br />
|[http://parts.igem.org/Part:BBa_K1159004 Part:BBa_K1159004]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3e7a 3e7a_A]<br />
|96%<br />
|1.593<br />
|[[File:TUM13_small_PP1.png|85px]]<br />
|-<br />
|GFP<br />
|[http://parts.igem.org/Part:BBa_K1159311 BBa_K1159311]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2WUR 2WUR]<br />
|98%<br />
|1.477<br />
|[[File:TUM13 small_GFP.png|85px]]<br />
|-<br />
|Glutathiontransferase / DDT Dehydrochlorinase<br />
|<partinfo>BBa_K620000</partinfo><br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3F6D 3F6D]<br />
|68%<br />
|1.155<br />
|[[File:TUM13 small_GST.png|85px]]<br />
|-<br />
|SERK-TM<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159305 BBa_K1159305]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2ks1 2ks1_B]<br />
|24%<br />
|0.233<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|TEV Protease<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=1Q31 1Q31]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|Streptavidin<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3RY2 3RY2]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|}<br />
<br />
====Results====<br />
The homology search showed that some of our effector proteins have very closely related proteins with a known structure. For example there are very similar protein structures available for the SypCatcher, PP1 and GFP each with identities of over 90%. Some other effector proteins such as XylE, Laccase or the DDT Dehydrochlorinase have less homologous proteins, whose structures still give good hints on structural questions. However there are also effectors where only badly matched structures are known, which can only be used as a very rough indication of the fold. The NanoLuc luciferase, which is a highly engineered protein derived from shrimps and was only published this year, is an example of a protein with no known structural homologue.<br><br />
The structures obtained here were used to design our experiments. A homology modeling for the Laccase was performed to determine whether it contains disulphide bridges. The resulting homologous structures were used as illustrations, as explained in one of our [https://2013.igem.org/Team:TU-Munich/Results/How_To How-Tos] about animated GIFs.<br />
<br />
==References:==<br />
[[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] MARGARET T. Boylan, M.T. and Quail, P.H. (1991). PhytochromeA overexpression inhibits hypocotyl elongation in transgenic ''Arabidopsis''. ''Proc. Natl. Acad. Sci.'' 88:10806-10810.<br><br />
<br />
[[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] Reski, R. (1998). Development, Genetics and Molecular Biology of Mosses. ''Bot. Acta'', 111:1-15.<br /><br />
<br />
[[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]] Lienhart, O. (2007). Untersuchungen zu einem Somatic-Embryogenesis-Receptor-like-Kinase-Homolog in ''Physcomitrella patens'' (Hedw.) B.S.G. PhD-thesis at Freiburg University<br />
<br />
[http://www.cbs.dtu.dk/services/SignalP/ SignalP41] Server<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]] Söding J, Biegert A, Lupas AN. (2005). The HHpred interactive server for protein homology detection and structure prediction. ''Nucleic Acids Res.'' 2005 Jul 1;33(Web Server issue):W244-8.<br><br />
<br />
<br />
<br />
<br />
<!-- Ende des Inhalts --><br />
</div><br />
</div><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Modeling/Protein_PredictionsTeam:TU-Munich/Modeling/Protein Predictions2013-10-29T03:33:00Z<p>FlorianA: /* Analysis of Receptor Sequences &ndash; Choosing the right template */</p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
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<div id="wikicontent"><br />
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<br />
==Prediction of Protein Structures and Functions==<br />
'''Structural properties''' of '''effector proteins''' are often essential for their function, so it is advantageous to know about them. It is for example necessary to know whether '''termini are accessible''' for protein fusion or whether the protein is '''only functional in a multimeric fold'''. For this reason a structure based search was performed in the [http://www.rcsb.org/pdb/home/home.do protein database]. As the number of identified structures is still limited, it is a promising attempt to look for homologous proteins whose crystal structures have been determined.<br />
<br />
==Analysis of Receptor Sequences &ndash; Choosing the right template ==<br />
<br />
For several purposes of our project, we needed a synthetic receptor enabling us to integrate proteins into the membrane in the desired orientation, i.e. to express protein-domains on the intracellular or extracellular side of the cell membrane. We investigated several different plant-receptors from the well characterized dicotyledon ''Arabidopsis thaliana'' and the moss ''Physcomitrella patens'', our chassis. The receptors from ''Arabidopsis thaliana'' have the advantage that their transgenic expression has successfully been demonstrated [[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] whereas the native receptors from ''Physcomitrella patens'' bear only a small risk of failing [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]].<br><br />
Due to the fact that there were many different available receptors, which we could have used as template for our synthetic receptor, we used bioinformatical methods to evaluate the suitability of these receptors. The following three examples ERF, FLS2 and SERK shown in table 1 resulted from this equation.<br />
<br />
{|cellspacing="0" border="1"<br />
|+ '''Table 1:''' Examined Receptors<br />
!Receptor<br />
!Organism<br />
!Length (aa)<br />
!Sequence reference<br />
!Literature reference<br />
|-<br />
|ERF<br />
|''A. thaliana''<br />
|1031<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_197548.1 NP_197548.1]<br />
|<br />
|-<br />
|FLS2<br />
|''A. thaliana''<br />
|1173<br />
|[http://www.ncbi.nlm.nih.gov/protein/NP_199445.1 NP_199445.1]<br />
|<br />
|-<br />
|SERK<br />
|''P. patens''<br />
|625<br />
|[http://www.ncbi.nlm.nih.gov/protein/XP_001759122.1 XP_001759122.1]<br />
|[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]<br />
|-<br />
|}<br />
<br><br />
===Prediction of Signal Peptides===<br />
[[File:TUM13 Modeling_Signal-P.png|thumb|right|350px| '''Figure 1:''' Prediction and analysis of signal peptides]]<br />
'''Introduction'''<br><br />
A first analysis was performed to identify signal-peptides, which are bound by the cellular signal recognition particle and lead to the translocation of the bound polypeptide into the endoplasmic reticulum. Afterwards the signal peptide is cleaved by a signal peptide peptidase at a specific site. The analysis for signal peptides was done by using the [http://www.cbs.dtu.dk/services/SignalP SignalP 4.1 Server]. <br><br />
<br><br />
'''Results'''<br><br />
The program was run for different receptors and will be illustrated for the three examples mentioned above (see fig. 1). <br><br />
The figure shows the N-terminal sequence of the receptors, together with three scores: <br><br />
(1) The C-Score (raw cleavage site score) in red. <br><br />
(2) The S-Score (signal peptide score) in green. <br><br />
(3) The Y-Score (combined cleavage site score) in blue.<br><br />
<br><br />
The C-Score shows the most probable cleavage site identified by the peptidase. It was possible to identify the most probable cleavage site for all shown receptors with ambiguous cleavage sites for the SERK-receptor. The amino acid with the highest C-score is predicted to be the first amino acid of the primary structure of the cleaved receptor. <br><br />
The S-Score was developed to identify amino acid sequences which appear in a signal peptide and others that belong to the matured receptor. The course of this parameter is high for the first 23-28 amino acids of all receptors, identifying these residues as signal peptides. The amino acid residue, which lies at the greatest decrease of the S-Score, is the predicted border between the N-terminal signal peptide and the receptor. <br><br />
The Y-Score results from the geometrical structure of the protein and the previously determined scoring parameters. It illustrates that the two first parameters show a good fit for the identification of the signal peptide in all three indicated receptors.<br><br />
<br><br />
'''Discussion'''<br><br />
Summarizing these parameters, it can be concluded that all three pictured receptors seem to contain a sequence acting as a signal peptide. For many of the predicted receptors in the genome of ''Physcomitrella patens'' the prediction did not yield a positive result. With respect to the signal peptide, all mentioned receptors would be suitable as a template for our synthetic receptor. The predicted data show that the SERK-Receptor is favorable for our application, because its signal peptide is statistically the best recognized one and bears the smallest risk of failure.<br />
<br />
<br><br />
<br><br />
<br />
===Prediction of Transmembrane Regions===<br />
[[File:TUM13 Modeling_TMHMM.png|thumb|right|350px| '''Figure 2:''' Prediction and analysis of transmembrane regions]]<br />
'''Introduction'''<br><br />
Additional to the identification of the signal peptide, it was very important to identify transmembrane regions within the receptors, because we wanted to use a type I receptor as a template that contains a N-terminal extracellular domain, a transmembrane domain and a C-terminal intracellular domain (see [https://2013.igem.org/Team:TU-Munich/Project/Localisation our localization page]). To analyze this issue, the prediction tool [http://www.cbs.dtu.dk/services/TMHMM TMHMM] was used for several different receptors. Again the most suitable receptors were ERK, FLS2 and SERK.<br><br />
<br><br />
'''Results'''<br><br />
The analysis yields a signal peptide and a single transmembrane domain for all three depicted receptors (see fig. 2). The estimated reliability of the predictions was equally good for all examined receptors, whereas the signal peptide was most reliably predicted for the SERK receptor. <br><br />
<br><br />
'''Discussion'''<br><br />
Focussing on the membrane topology point of view, all the investigated receptors would be suitable blue prints for our synthetic receptor. As the SERK-Receptor yields the best prediction, it was chosen as the favorable template. Another reason to choose the SERK-Receptor was that it is derived from ''Physcomitrella patens''. The only problem, concerning this prediction, is that the N-terminus of this receptor is predicted to be extracellular. The falsification of this prediction was simple, because the SERK receptor contains a C-terminal kinase-domain, which is known to be involved in signal transduction.<br />
<br><br />
<br><br />
<br />
===Choice of the SERK Receptor===<br />
Finally we decided to use the SERK receptor as a template to generate our synthetic receptor. The final receptor was designed in RFC[25] standard, which allows in frame protein fusions. The final constructs were designed containing the SERK signal peptide ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]), an extracellularly located effector protein, the transmembrane domain of the SERK receptor ([http://parts.igem.org/Part:BBa_K1159305 BBa_K1159305]), a short linker and a GFP, to investigate the cellular localization of our receptor with the aid of fluorescence microscopy.<br />
<br />
==Searching for Homologous Structures using HHpred==<br />
<br />
The search for homologous structures was performed by using the freely accessible web server [http://toolkit.tuebingen.mpg.de/hhpred HHpred] [[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]]. The amino acid sequences for the BioBricks were translated into amino acid sequences using the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] and was then inserted into the the search field. The results for all proteins investigated in our project are shown in table 2. <br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 2:''' Predicted Structures<br />
!Protein<br />
!BioBrick<br />
!PDB-code<br />
!Identity<br />
!Similarity<br />
!Structure<br />
|-<br />
|XylE<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K147002 BBa_K147002]<br />
| [http://www.rcsb.org/pdb/explore.do?structureId=3hpy 3hpy_A]<br />
|50%<br />
|0.939<br />
|[[File:TUM13 small_XylE.png|85px]]<br />
|-<br />
|Laccase<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159002 BBa_K1159002]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2wsd 2wsd_A]<br />
|68%<br />
|1.223<br />
|[[File:TUM13 small_Laccase.png|85px]]<br />
|-<br />
|NanoLuc<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159001 BBa_K1159001]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3ppt 3ppt_A]<br />
|21%<br />
|0.359<br />
|[[File:TUM13 small_NanoLuc.png|85px]]<br />
|-<br />
|EreB<br />
|[http://parts.igem.org/Part:BBa_K1159000 BBa_K1159000]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3b55 3b55_A]<br />
|19%<br />
|0.318<br />
|[[File:TUM13_small_EreBx.png|85px]]<br />
|-<br />
|Spycatcher<br />
|[http://parts.igem.org/Part:BBa_K1159200 BBa_K1159200]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2x5p 2x5p_A]<br />
|97%<br />
|1.298<br />
|[[File:TUM13 small_SpyCatcher.png|85px]]<br />
|-<br />
|PP1<br />
|[http://parts.igem.org/Part:BBa_K1159004 Part:BBa_K1159004]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3e7a 3e7a_A]<br />
|96%<br />
|1.593<br />
|[[File:TUM13_small_PP1.png|85px]]<br />
|-<br />
|GFP<br />
|[http://parts.igem.org/Part:BBa_K1159311 BBa_K1159311]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2WUR 2WUR]<br />
|98%<br />
|1.477<br />
|[[File:TUM13 small_GFP.png|85px]]<br />
|-<br />
|Glutathiontransferase / DDT Dehydrochlorinase<br />
|<partinfo>BBa_K620000</partinfo><br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3F6D 3F6D]<br />
|68%<br />
|1.155<br />
|[[File:TUM13 small_GST.png|85px]]<br />
|-<br />
|SERK-TM<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159305 BBa_K1159305]<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=2ks1 2ks1_B]<br />
|24%<br />
|0.233<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|TEV Protease<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=1Q31 1Q31]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|Streptavidin<br />
|Commercial reagent<br />
|[http://www.rcsb.org/pdb/explore.do?structureId=3RY2 3RY2]<br />
|n.d.<br />
|n.d.<br />
|[[File:Blanko2.png|85px]]<br />
|-<br />
|}<br />
<br />
====Results====<br />
The homology search showed that some of our effector proteins have very closely related proteins with a known structure. For example there are very similar protein structures available for the SypCatcher, PP1 and GFP each with identities of over 90%. Some other effector proteins such as XylE, Laccase or the DDT Dehydrochlorinase have less homologous proteins, whose structures still give good hints on structural questions. However there are also effectors where only badly matched structures are known, which can only be used as a very rough indication of the fold. The NanoLuc luciferase, which is a highly engineered protein derived from shrimps and was only published this year, is an example of a protein with no known structural homologue.<br><br />
The structures obtained here were used to design our experiments. A homology modeling for the Laccase was performed to determine whether it contains disulphide bridges. The resulting homologous structures were used as illustrations, as explained in one of our [https://2013.igem.org/Team:TU-Munich/Results/How_To How-Tos] about animated GIFs.<br />
<br />
==References:==<br />
[[http://www.pnas.org/content/88/23/10806.full.pdf Quail et al., 1991]] MARGARET T. Boylan, M.T. and Quail, P.H. (1991). PhytochromeA overexpression inhibits hypocotyl elongation in transgenic ''Arabidopsis''. ''Proc. Natl. Acad. Sci.'' 88:10806-10810.<br><br />
<br />
[[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] Reski, R. (1998). Development, Genetics and Molecular Biology of Mosses. ''Bot. Acta'', 111:1-15.<br /><br />
<br />
[[http://www.freidok.uni-freiburg.de/volltexte/5390/pdf/Lienhart_Dissertation_2008.pdf Lienhart, 2007]] Lienhart, O. (2007). Untersuchungen zu einem Somatic-Embryogenesis-Receptor-like-Kinase-Homolog in ''Physcomitrella patens'' (Hedw.) B.S.G. PhD-thesis at Freiburg University<br />
<br />
[http://www.cbs.dtu.dk/services/SignalP/ SignalP41] Server<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/15980461 Söding et al., 2005]] Söding J, Biegert A, Lupas AN. (2005). The HHpred interactive server for protein homology detection and structure prediction. ''Nucleic Acids Res.'' 2005 Jul 1;33(Web Server issue):W244-8.<br><br />
<br />
<br />
<br />
<br />
<!-- Ende des Inhalts --><br />
</div><br />
</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/KillSwitchTeam:TU-Munich/Results/KillSwitch2013-10-29T03:26:10Z<p>FlorianA: /* Photosensitivity */</p>
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<br />
==Kill Switch==<br />
[[File:TUM13_Moostot_(2).png|thumb|left|300px| '''Figure 1''': Dead Kill Switch moss protonema]]<br />
<br />
The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. For details on its design and function see [https://2013.igem.org/Team:TU-Munich/Project/Killswitch here]. We were very excited to start experimenting with the killswitch moss. <br />
<br />
However, when we opened the redlight filter foil, we found both the PIF3 and the PIF6 version of the allegedly transgenic moss lines were dead. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach and produce live Kill Switch moss lines.<br />
<br />
==Transformation of the large DNA constructs==<br />
<br />
The Kill Switch DNA constructs are very large and even exceed the Part Registry´s frame (see Figure 2). For the PEG-mediated moss [https://2013.igem.org/Team:TU-Munich/Notebook/Methods#Transformation transformation], we used plasmids linearized with EcoRI, but contrary to the targeted gene transfer described by [http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2003], our constructs contained no flanking homologuos regions. Our random integration transformation worked successfully for our other contructs, whereas the large Kill Switch construct might need further inquiry for a suitable integration site. Due to its size, the construct might be less stable and require sensitive handling. These factors could have lead to a failed transformation, in which case the moss would have died through the G418 selection process.<br />
<br />
[[File:TUM13_PartsRegistryKillSwitch.png|thumb|center|650px| '''Figure 2''': [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118]]<br />
<br />
We weren´t yet able to express the Kill Switch in ''E.coli'', which is most likely due to codon usage discrepancy. While ''Physcomitrella patens'' is not very dicriminating regarding codon usage, the function and expression of proteins designed for ''Physco'' is not guaranteed to work in ''E.coli''.<br />
<br />
==Level of promoter activity==<br />
<br />
[[File:PBI_376_f1.gif|thumb|right|300px| '''Figure 4''': Comparative expression performance of different constitutive mammalian and plant promoters in Physcomitrella patens, [[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]]]]<br />
<br />
We used the Actin 5 promotor from ''Physcomitrella patens'' [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306], which is the strongest known Physco promotor (see Figure 3). The very high intensity of expression might lead to problems concerning the vitality of the transgenic moss. <br />
<br />
Because the Kill Switch encodes for the very effective thermonuclease, overexpression might lead to loss of vitality in the moss regardless of its fixation to the cell membrane. Equipping the Kill Switch with a promotor for a lower level of expression, such as the CMV promotor or the SV40 promotor, could maybe lead to a higher survival rate of successfully transformed moss.<br />
<br />
==Photosensitivity==<br />
[[File:TUM13_Moosfolie.jpg|thumb|right|250px| '''Figure 4''': Photosensitive moss protected by foil wrapping]]<br />
The programmed cell death triggered through the Kill Switch is induced through the red light fraction of the spectrum of visible light. It is therefore crucial to shield transformed moss plants from light! We used a layer combination green and blue foil that was wrapped arouns the protoplast 6-wells right after transformation, (see Figure 4). <br />
<br />
[[File:TUM13_Foto_Labpics2.jpg|thumb|right|250px| '''Figure 5''': Working with the photosensitive protoplasts]]<br />
To start the selection process, the still vital regenerated moss cells were transfered onto G418 agar plates at night, lighting the workspace with a lamp wrapped up in the previously described foils, (see Figure 5). <br />
<br />
Since the moss survived until this point, we can conclude that the light the foil allows to pass is sufficient for the moss to grow. Its early death is therefore not due to insufficient light supply. Given the precautions we met in the handling process, accidental contact with unfiltered light can be ruled out as a reason. <br />
<br />
However, it is not guaranteed that the foil layers are sufficient to protect the moss from red light and of course we could not yet determine which wavelenghts define the limits of the spectrum the moss can tolerate and which intensity of light inside the spectrum of interest is needed to trigger the TEV protease fusion.<br />
<br />
==References==<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Feb;7(2):210<br />
<br />
[[http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2004]] An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, ''Physcomitrella patens'', Curr Genet (2004) 44: 339–347<br />
<br />
<div class="visualClear"></div><br />
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<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/KillSwitchTeam:TU-Munich/Results/KillSwitch2013-10-29T03:25:48Z<p>FlorianA: /* Level of promoter activity */</p>
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<br />
==Kill Switch==<br />
[[File:TUM13_Moostot_(2).png|thumb|left|300px| '''Figure 1''': Dead Kill Switch moss protonema]]<br />
<br />
The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. For details on its design and function see [https://2013.igem.org/Team:TU-Munich/Project/Killswitch here]. We were very excited to start experimenting with the killswitch moss. <br />
<br />
However, when we opened the redlight filter foil, we found both the PIF3 and the PIF6 version of the allegedly transgenic moss lines were dead. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach and produce live Kill Switch moss lines.<br />
<br />
==Transformation of the large DNA constructs==<br />
<br />
The Kill Switch DNA constructs are very large and even exceed the Part Registry´s frame (see Figure 2). For the PEG-mediated moss [https://2013.igem.org/Team:TU-Munich/Notebook/Methods#Transformation transformation], we used plasmids linearized with EcoRI, but contrary to the targeted gene transfer described by [http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2003], our constructs contained no flanking homologuos regions. Our random integration transformation worked successfully for our other contructs, whereas the large Kill Switch construct might need further inquiry for a suitable integration site. Due to its size, the construct might be less stable and require sensitive handling. These factors could have lead to a failed transformation, in which case the moss would have died through the G418 selection process.<br />
<br />
[[File:TUM13_PartsRegistryKillSwitch.png|thumb|center|650px| '''Figure 2''': [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118]]<br />
<br />
We weren´t yet able to express the Kill Switch in ''E.coli'', which is most likely due to codon usage discrepancy. While ''Physcomitrella patens'' is not very dicriminating regarding codon usage, the function and expression of proteins designed for ''Physco'' is not guaranteed to work in ''E.coli''.<br />
<br />
==Level of promoter activity==<br />
<br />
[[File:PBI_376_f1.gif|thumb|right|300px| '''Figure 4''': Comparative expression performance of different constitutive mammalian and plant promoters in Physcomitrella patens, [[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]]]]<br />
<br />
We used the Actin 5 promotor from ''Physcomitrella patens'' [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306], which is the strongest known Physco promotor (see Figure 3). The very high intensity of expression might lead to problems concerning the vitality of the transgenic moss. <br />
<br />
Because the Kill Switch encodes for the very effective thermonuclease, overexpression might lead to loss of vitality in the moss regardless of its fixation to the cell membrane. Equipping the Kill Switch with a promotor for a lower level of expression, such as the CMV promotor or the SV40 promotor, could maybe lead to a higher survival rate of successfully transformed moss.<br />
<br />
==Photosensitivity==<br />
[[File:TUM13_Moosfolie.jpg|thumb|right|250px| '''Figure 5''': Photosensitive moss protected by foil wrapping]]<br />
The programmed cell death triggered through the Kill Switch is induced through the red light fraction of the spectrum of visible light. It is therefore crucial to shield transformed moss plants from light! We used a layer combination green and blue foil that was wrapped arouns the protoplast 6-wells right after transformation, (see Figure 5). <br />
<br />
[[File:TUM13_Foto_Labpics2.jpg|thumb|right|250px| '''Figure 6''': Working with the photosensitive protoplasts]]<br />
To start the selection process, the still vital regenerated moss cells were transfered onto G418 agar plates at night, lighting the workspace with a lamp wrapped up in the previously described foils, (see Figure 6). <br />
<br />
Since the moss survived until this point, we can conclude that the light the foil allows to pass is sufficient for the moss to grow. Its early death is therefore not due to insufficient light supply. Given the precautions we met in the handling process, accidental contact with unfiltered light can be ruled out as a reason. <br />
<br />
However, it is not guaranteed that the foil layers are sufficient to protect the moss from red light and of course we could not yet determine which wavelenghts define the limits of the spectrum the moss can tolerate and which intensity of light inside the spectrum of interest is needed to trigger the TEV protease fusion.<br />
<br />
==References==<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Feb;7(2):210<br />
<br />
[[http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2004]] An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, ''Physcomitrella patens'', Curr Genet (2004) 44: 339–347<br />
<br />
<div class="visualClear"></div><br />
<!-- End of content --><br />
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</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:23:31Z<p>FlorianA: /* Tolerance to relevant environmental pollutants and toxins */</p>
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<br />
== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 2''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 3''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 4''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/TUM13_Foto_Freiburgvisit1.jpg/350px-TUM13_Foto_Freiburgvisit1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/41/TUM13_Foto_Freiburgvisit2.jpg/350px-TUM13_Foto_Freiburgvisit2.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_Foto_Freiburgvisit3.jpg/350px-TUM13_Foto_Freiburgvisit3.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/25/TUM13_Foto_Freiburgvisit4.jpg/350px-TUM13_Foto_Freiburgvisit4.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/13/TUM13_Foto_Freiburgvisit5.jpg/350px-TUM13_Foto_Freiburgvisit5.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/50/TUM13_Foto_Freiburgvisit6.jpg/350px-TUM13_Foto_Freiburgvisit6.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_Foto_Freiburgvisit7.jpg/350px-TUM13_Foto_Freiburgvisit7.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/46/TUM13_Foto_Freiburgvisit8.jpg/350px-TUM13_Foto_Freiburgvisit8.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/df/TUM13_Foto_Freiburgvisit9.jpg/350px-TUM13_Foto_Freiburgvisit9.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/2e/TUM13_Foto_Freiburgvisit10.jpg/350px-TUM13_Foto_Freiburgvisit10.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/26/TUM13_Foto_Freiburgvisit11.jpg/350px-TUM13_Foto_Freiburgvisit11.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/de/TUM13_Foto_Freiburgvisit12.jpg/350px-TUM13_Foto_Freiburgvisit12.jpg" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/e6/TUM13_Foto_Freiburgvisit15.jpg/350px-TUM13_Foto_Freiburgvisit15.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/70/TUM13_Foto_Freiburgvisit16.jpg/350px-TUM13_Foto_Freiburgvisit16.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/8e/TUM13_Foto_Freiburgvisit17.jpg/350px-TUM13_Foto_Freiburgvisit17.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/b9/TUM13_Foto_Freiburgvisit18.jpg/350px-TUM13_Foto_Freiburgvisit18.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/94/TUM13_Foto_Freiburgvisit19.jpg/350px-TUM13_Foto_Freiburgvisit19.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/fc/TUM13_Foto_Freiburgvisit20.jpg/350px-TUM13_Foto_Freiburgvisit20.jpg" /></li><br />
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<br />
We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/be/TUM13_surface2.png/350px-TUM13_surface2.png" title="<i>Physco</i> on felt" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/89/TUM13_surface4.png/350px-TUM13_surface4.png" title="<i>Physco</i> on agar" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0e/TUM13_surface3.png/350px-TUM13_surface3.png" title="<i>Physco</i> on metal grid" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_surface1.png/350px-TUM13_surface1.png" title="<i>Physco</i> on cotton cloth" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_surface7.png/350px-TUM13_surface7.png" title="<i>Physco</i> on microfiber" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/9d/TUM13_surface5.png/350px-TUM13_surface5.png" title="Comparison of materials"/></li><br />
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<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
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<br />
In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 5''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 6:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 7:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 2''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:23:02Z<p>FlorianA: /* Tolerance to relevant environmental pollutants and toxins */</p>
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== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 2''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 3''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 4''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/TUM13_Foto_Freiburgvisit1.jpg/350px-TUM13_Foto_Freiburgvisit1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/41/TUM13_Foto_Freiburgvisit2.jpg/350px-TUM13_Foto_Freiburgvisit2.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_Foto_Freiburgvisit3.jpg/350px-TUM13_Foto_Freiburgvisit3.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/25/TUM13_Foto_Freiburgvisit4.jpg/350px-TUM13_Foto_Freiburgvisit4.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/13/TUM13_Foto_Freiburgvisit5.jpg/350px-TUM13_Foto_Freiburgvisit5.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/50/TUM13_Foto_Freiburgvisit6.jpg/350px-TUM13_Foto_Freiburgvisit6.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_Foto_Freiburgvisit7.jpg/350px-TUM13_Foto_Freiburgvisit7.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/46/TUM13_Foto_Freiburgvisit8.jpg/350px-TUM13_Foto_Freiburgvisit8.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/df/TUM13_Foto_Freiburgvisit9.jpg/350px-TUM13_Foto_Freiburgvisit9.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/2e/TUM13_Foto_Freiburgvisit10.jpg/350px-TUM13_Foto_Freiburgvisit10.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/26/TUM13_Foto_Freiburgvisit11.jpg/350px-TUM13_Foto_Freiburgvisit11.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/de/TUM13_Foto_Freiburgvisit12.jpg/350px-TUM13_Foto_Freiburgvisit12.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit13.jpg/350px-TUM13_Foto_Freiburgvisit13.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/ec/TUM13_Foto_Freiburgvisit14.jpg/350px-TUM13_Foto_Freiburgvisit14.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/e6/TUM13_Foto_Freiburgvisit15.jpg/350px-TUM13_Foto_Freiburgvisit15.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/70/TUM13_Foto_Freiburgvisit16.jpg/350px-TUM13_Foto_Freiburgvisit16.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/8e/TUM13_Foto_Freiburgvisit17.jpg/350px-TUM13_Foto_Freiburgvisit17.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/b9/TUM13_Foto_Freiburgvisit18.jpg/350px-TUM13_Foto_Freiburgvisit18.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/94/TUM13_Foto_Freiburgvisit19.jpg/350px-TUM13_Foto_Freiburgvisit19.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/fc/TUM13_Foto_Freiburgvisit20.jpg/350px-TUM13_Foto_Freiburgvisit20.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/54/TUM13_Foto_Freiburgvisit21.jpg/350px-TUM13_Foto_Freiburgvisit21.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/5a/TUM13_Foto_Freiburgvisit22.jpg/350px-TUM13_Foto_Freiburgvisit22.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/23/TUM13_Foto_Freiburgvisit23.jpg/350px-TUM13_Foto_Freiburgvisit23.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/91/TUM13_Foto_Freiburgvisit24.jpg/350px-TUM13_Foto_Freiburgvisit24.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/81/TUM13_Foto_Freiburgvisit25.jpg/350px-TUM13_Foto_Freiburgvisit25.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f9/TUM13_Foto_Freiburgvisit26.jpg/350px-TUM13_Foto_Freiburgvisit26.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit27.jpg/350px-TUM13_Foto_Freiburgvisit27.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f2/TUM13_Foto_Freiburgvisit28.jpg/350px-TUM13_Foto_Freiburgvisit28.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0d/TUM13_Foto_Freiburgvisit29.jpg/350px-TUM13_Foto_Freiburgvisit29.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/a7/TUM13_Foto_Freiburgvisit30.jpg/350px-TUM13_Foto_Freiburgvisit30.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/ab/TUM13_Foto_Freiburgvisit31.jpg/350px-TUM13_Foto_Freiburgvisit31.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3d/TUM13_Foto_Freiburgvisit32.jpg/350px-TUM13_Foto_Freiburgvisit32.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/06/TUM13_Foto_Freiburgvisit33.jpg/350px-TUM13_Foto_Freiburgvisit33.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/12/TUM13_Foto_Freiburgvisit34.jpg/350px-TUM13_Foto_Freiburgvisit34.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/31/TUM13_Foto_Freiburgvisit35.jpg/350px-TUM13_Foto_Freiburgvisit35.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/16/TUM13_Foto_Freiburgvisit36.jpg/350px-TUM13_Foto_Freiburgvisit36.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/95/TUM13_Foto_Freiburgvisit37.jpg/350px-TUM13_Foto_Freiburgvisit37.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/ac/TUM13_Foto_Freiburgvisit38.jpg/350px-TUM13_Foto_Freiburgvisit38.jpg" /></li><br />
</ul><br />
</div><br />
</html><br />
<br />
We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
<!---<br />
[[File:TUM13_surface1.png|thumb|350px]]<br />
[[File:TUM13_surface2.png|thumb|350px]]<br />
[[File:TUM13_surface3.png|thumb|350px]]<br />
[[File:TUM13_surface4.png|thumb|350px]]<br />
[[File:TUM13_surface5.png|thumb|350px]]<br />
[[File:TUM13_surface6.png|thumb|350px]]<br />
[[File:TUM13_surface7.png|thumb|350px]]<br />
---><br />
<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/be/TUM13_surface2.png/350px-TUM13_surface2.png" title="<i>Physco</i> on felt" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/89/TUM13_surface4.png/350px-TUM13_surface4.png" title="<i>Physco</i> on agar" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0e/TUM13_surface3.png/350px-TUM13_surface3.png" title="<i>Physco</i> on metal grid" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_surface1.png/350px-TUM13_surface1.png" title="<i>Physco</i> on cotton cloth" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_surface7.png/350px-TUM13_surface7.png" title="<i>Physco</i> on microfiber" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/9d/TUM13_surface5.png/350px-TUM13_surface5.png" title="Comparison of materials"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/54/TUM13_surface6.png/350px-TUM13_surface6.png" title="Comparison of materials"/></li><br />
</ul><br />
</div><br />
</html><br />
<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/77/TUM13_wave_reactor1.png/350px-TUM13_wave_reactor1.png" title="Wave reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
</ul><br />
</div><br />
</html><br />
<br />
In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 5''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 6:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 8:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 2''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:22:19Z<p>FlorianA: /* Determination of growth rates for different liquid culture forms */</p>
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== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 2''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 3''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 4''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
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<br />
We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
<!---<br />
[[File:TUM13_surface1.png|thumb|350px]]<br />
[[File:TUM13_surface2.png|thumb|350px]]<br />
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[[File:TUM13_surface4.png|thumb|350px]]<br />
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---><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/be/TUM13_surface2.png/350px-TUM13_surface2.png" title="<i>Physco</i> on felt" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/89/TUM13_surface4.png/350px-TUM13_surface4.png" title="<i>Physco</i> on agar" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0e/TUM13_surface3.png/350px-TUM13_surface3.png" title="<i>Physco</i> on metal grid" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_surface1.png/350px-TUM13_surface1.png" title="<i>Physco</i> on cotton cloth" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_surface7.png/350px-TUM13_surface7.png" title="<i>Physco</i> on microfiber" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/9d/TUM13_surface5.png/350px-TUM13_surface5.png" title="Comparison of materials"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/54/TUM13_surface6.png/350px-TUM13_surface6.png" title="Comparison of materials"/></li><br />
</ul><br />
</div><br />
</html><br />
<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/77/TUM13_wave_reactor1.png/350px-TUM13_wave_reactor1.png" title="Wave reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
</ul><br />
</div><br />
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<br />
In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 5''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 6:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 3:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 1''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:21:19Z<p>FlorianA: /* 2. Preparation of linear DNA */</p>
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<br />
== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 2''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 3''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 4''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
<html><br />
<div class="box-center"><br />
<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/TUM13_Foto_Freiburgvisit1.jpg/350px-TUM13_Foto_Freiburgvisit1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/41/TUM13_Foto_Freiburgvisit2.jpg/350px-TUM13_Foto_Freiburgvisit2.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_Foto_Freiburgvisit3.jpg/350px-TUM13_Foto_Freiburgvisit3.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/25/TUM13_Foto_Freiburgvisit4.jpg/350px-TUM13_Foto_Freiburgvisit4.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/13/TUM13_Foto_Freiburgvisit5.jpg/350px-TUM13_Foto_Freiburgvisit5.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/50/TUM13_Foto_Freiburgvisit6.jpg/350px-TUM13_Foto_Freiburgvisit6.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_Foto_Freiburgvisit7.jpg/350px-TUM13_Foto_Freiburgvisit7.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/46/TUM13_Foto_Freiburgvisit8.jpg/350px-TUM13_Foto_Freiburgvisit8.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/df/TUM13_Foto_Freiburgvisit9.jpg/350px-TUM13_Foto_Freiburgvisit9.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/2e/TUM13_Foto_Freiburgvisit10.jpg/350px-TUM13_Foto_Freiburgvisit10.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/26/TUM13_Foto_Freiburgvisit11.jpg/350px-TUM13_Foto_Freiburgvisit11.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/de/TUM13_Foto_Freiburgvisit12.jpg/350px-TUM13_Foto_Freiburgvisit12.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit13.jpg/350px-TUM13_Foto_Freiburgvisit13.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/ec/TUM13_Foto_Freiburgvisit14.jpg/350px-TUM13_Foto_Freiburgvisit14.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/e6/TUM13_Foto_Freiburgvisit15.jpg/350px-TUM13_Foto_Freiburgvisit15.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/70/TUM13_Foto_Freiburgvisit16.jpg/350px-TUM13_Foto_Freiburgvisit16.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/8e/TUM13_Foto_Freiburgvisit17.jpg/350px-TUM13_Foto_Freiburgvisit17.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/b9/TUM13_Foto_Freiburgvisit18.jpg/350px-TUM13_Foto_Freiburgvisit18.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/94/TUM13_Foto_Freiburgvisit19.jpg/350px-TUM13_Foto_Freiburgvisit19.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/fc/TUM13_Foto_Freiburgvisit20.jpg/350px-TUM13_Foto_Freiburgvisit20.jpg" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/23/TUM13_Foto_Freiburgvisit23.jpg/350px-TUM13_Foto_Freiburgvisit23.jpg" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f9/TUM13_Foto_Freiburgvisit26.jpg/350px-TUM13_Foto_Freiburgvisit26.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit27.jpg/350px-TUM13_Foto_Freiburgvisit27.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f2/TUM13_Foto_Freiburgvisit28.jpg/350px-TUM13_Foto_Freiburgvisit28.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0d/TUM13_Foto_Freiburgvisit29.jpg/350px-TUM13_Foto_Freiburgvisit29.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/a7/TUM13_Foto_Freiburgvisit30.jpg/350px-TUM13_Foto_Freiburgvisit30.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/ab/TUM13_Foto_Freiburgvisit31.jpg/350px-TUM13_Foto_Freiburgvisit31.jpg" /></li><br />
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<br />
We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/be/TUM13_surface2.png/350px-TUM13_surface2.png" title="<i>Physco</i> on felt" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/89/TUM13_surface4.png/350px-TUM13_surface4.png" title="<i>Physco</i> on agar" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0e/TUM13_surface3.png/350px-TUM13_surface3.png" title="<i>Physco</i> on metal grid" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_surface1.png/350px-TUM13_surface1.png" title="<i>Physco</i> on cotton cloth" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_surface7.png/350px-TUM13_surface7.png" title="<i>Physco</i> on microfiber" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/9d/TUM13_surface5.png/350px-TUM13_surface5.png" title="Comparison of materials"/></li><br />
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<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
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<br />
In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 1''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 2:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 3:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 1''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:20:51Z<p>FlorianA: /* 1. Generation of expression constructs */</p>
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== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 5''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 6''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 7''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
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<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/TUM13_Foto_Freiburgvisit1.jpg/350px-TUM13_Foto_Freiburgvisit1.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/41/TUM13_Foto_Freiburgvisit2.jpg/350px-TUM13_Foto_Freiburgvisit2.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_Foto_Freiburgvisit3.jpg/350px-TUM13_Foto_Freiburgvisit3.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/25/TUM13_Foto_Freiburgvisit4.jpg/350px-TUM13_Foto_Freiburgvisit4.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/13/TUM13_Foto_Freiburgvisit5.jpg/350px-TUM13_Foto_Freiburgvisit5.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/50/TUM13_Foto_Freiburgvisit6.jpg/350px-TUM13_Foto_Freiburgvisit6.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_Foto_Freiburgvisit7.jpg/350px-TUM13_Foto_Freiburgvisit7.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/46/TUM13_Foto_Freiburgvisit8.jpg/350px-TUM13_Foto_Freiburgvisit8.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/df/TUM13_Foto_Freiburgvisit9.jpg/350px-TUM13_Foto_Freiburgvisit9.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/2e/TUM13_Foto_Freiburgvisit10.jpg/350px-TUM13_Foto_Freiburgvisit10.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/26/TUM13_Foto_Freiburgvisit11.jpg/350px-TUM13_Foto_Freiburgvisit11.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/de/TUM13_Foto_Freiburgvisit12.jpg/350px-TUM13_Foto_Freiburgvisit12.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit13.jpg/350px-TUM13_Foto_Freiburgvisit13.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/ec/TUM13_Foto_Freiburgvisit14.jpg/350px-TUM13_Foto_Freiburgvisit14.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/e/e6/TUM13_Foto_Freiburgvisit15.jpg/350px-TUM13_Foto_Freiburgvisit15.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/70/TUM13_Foto_Freiburgvisit16.jpg/350px-TUM13_Foto_Freiburgvisit16.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/8e/TUM13_Foto_Freiburgvisit17.jpg/350px-TUM13_Foto_Freiburgvisit17.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/b9/TUM13_Foto_Freiburgvisit18.jpg/350px-TUM13_Foto_Freiburgvisit18.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/94/TUM13_Foto_Freiburgvisit19.jpg/350px-TUM13_Foto_Freiburgvisit19.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/fc/TUM13_Foto_Freiburgvisit20.jpg/350px-TUM13_Foto_Freiburgvisit20.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/54/TUM13_Foto_Freiburgvisit21.jpg/350px-TUM13_Foto_Freiburgvisit21.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/5a/TUM13_Foto_Freiburgvisit22.jpg/350px-TUM13_Foto_Freiburgvisit22.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/23/TUM13_Foto_Freiburgvisit23.jpg/350px-TUM13_Foto_Freiburgvisit23.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/91/TUM13_Foto_Freiburgvisit24.jpg/350px-TUM13_Foto_Freiburgvisit24.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/81/TUM13_Foto_Freiburgvisit25.jpg/350px-TUM13_Foto_Freiburgvisit25.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f9/TUM13_Foto_Freiburgvisit26.jpg/350px-TUM13_Foto_Freiburgvisit26.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3b/TUM13_Foto_Freiburgvisit27.jpg/350px-TUM13_Foto_Freiburgvisit27.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/f/f2/TUM13_Foto_Freiburgvisit28.jpg/350px-TUM13_Foto_Freiburgvisit28.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0d/TUM13_Foto_Freiburgvisit29.jpg/350px-TUM13_Foto_Freiburgvisit29.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/a7/TUM13_Foto_Freiburgvisit30.jpg/350px-TUM13_Foto_Freiburgvisit30.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/ab/TUM13_Foto_Freiburgvisit31.jpg/350px-TUM13_Foto_Freiburgvisit31.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/3d/TUM13_Foto_Freiburgvisit32.jpg/350px-TUM13_Foto_Freiburgvisit32.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/06/TUM13_Foto_Freiburgvisit33.jpg/350px-TUM13_Foto_Freiburgvisit33.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/12/TUM13_Foto_Freiburgvisit34.jpg/350px-TUM13_Foto_Freiburgvisit34.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/3/31/TUM13_Foto_Freiburgvisit35.jpg/350px-TUM13_Foto_Freiburgvisit35.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/1/16/TUM13_Foto_Freiburgvisit36.jpg/350px-TUM13_Foto_Freiburgvisit36.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/95/TUM13_Foto_Freiburgvisit37.jpg/350px-TUM13_Foto_Freiburgvisit37.jpg" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/a/ac/TUM13_Foto_Freiburgvisit38.jpg/350px-TUM13_Foto_Freiburgvisit38.jpg" /></li><br />
</ul><br />
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<br />
We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
<!---<br />
[[File:TUM13_surface1.png|thumb|350px]]<br />
[[File:TUM13_surface2.png|thumb|350px]]<br />
[[File:TUM13_surface3.png|thumb|350px]]<br />
[[File:TUM13_surface4.png|thumb|350px]]<br />
[[File:TUM13_surface5.png|thumb|350px]]<br />
[[File:TUM13_surface6.png|thumb|350px]]<br />
[[File:TUM13_surface7.png|thumb|350px]]<br />
---><br />
<br />
<html><br />
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<ul class="bxgallery"><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/be/TUM13_surface2.png/350px-TUM13_surface2.png" title="<i>Physco</i> on felt" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/89/TUM13_surface4.png/350px-TUM13_surface4.png" title="<i>Physco</i> on agar" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/0/0e/TUM13_surface3.png/350px-TUM13_surface3.png" title="<i>Physco</i> on metal grid" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/d/d8/TUM13_surface1.png/350px-TUM13_surface1.png" title="<i>Physco</i> on cotton cloth" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/2/27/TUM13_surface7.png/350px-TUM13_surface7.png" title="<i>Physco</i> on microfiber" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/9/9d/TUM13_surface5.png/350px-TUM13_surface5.png" title="Comparison of materials"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/5/54/TUM13_surface6.png/350px-TUM13_surface6.png" title="Comparison of materials"/></li><br />
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<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
<html><br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/77/TUM13_wave_reactor1.png/350px-TUM13_wave_reactor1.png" title="Wave reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
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<br />
In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 1''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 2:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 3:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 1''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/MossTeam:TU-Munich/Results/Moss2013-10-29T03:20:24Z<p>FlorianA: /* Creation of transgenic Physcomitrella patens plants */</p>
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== Creation of transgenic ''Physcomitrella patens'' plants ==<br />
<br />
[[File:TUM13_Workflow_Transformation.png|thumb|center|910px|'''Figure 1:''' Work flow for the generation of transgenic moss.]]<br />
<br />
===1. Generation of expression constructs===<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 2:''' Transgenic ''Physcomitrella patens'' plants<br />
!Number<br />
!Construct name (abbreviation)<br />
!BioBrick<br />
!Successful Transformation?<br />
|-<br />
|PF-1<br />
|GFP (GFP-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-2<br />
|Igk-GFP (GFP-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159304 BBa_K1159304] + [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159311 BBa_K1159311]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-3<br />
|NanoLuciferase (nLuc-cyt)<br />
|<partinfo>BBa_K1159001</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-4<br />
|Igk-NanoLuciferase (nLuc-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159006 BBa_K1159006]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-5<br />
|SERK-NanoLuciferase (SERK-nLuc)<br />
|<partinfo>BBa_K1159010</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-6<br />
|NanoLuciferase-Receptor (nLuc-rec)<br />
|<partinfo>BBa_K1159015</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-7<br />
|Erythromycinesterase (EreB-cyt)<br />
|<partinfo>BBa_K1159000</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-8<br />
|Erythromycinesterase-Receptor (EreB-rec)<br />
|<partinfo>BBa_K1159014</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-9<br />
|Ig Kappa Erythromycinesterase (EreB-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159005 BBa_K1159005]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-10<br />
|Laccase-Receptor (Lac-rec)<br />
|<partinfo>BBa_K1159016</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-11<br />
|Ig Kappa Laccase (Lac-sec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159007 BBa_K1159007]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-12<br />
|Catecholdioxygenase (XylE-cyt)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159012 BBa_K1159012]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-13<br />
|DDT-dehydrochlorinase (GST-cyt)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-14<br />
|PP1-Receptor (PP1-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159019 BBa_K1159019]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-15<br />
|FluA-Receptor (FluA-rec)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159017 BBa_K1159017]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-16<br />
|Stressinducible_Promoter-RFP (Stress)<br />
|<partinfo>BBa_E0040</partinfo><br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-17<br />
|SpyCatcher-Receptor:Spytag-nLuc (Catcher:Tag-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159212 BBa_K1159212]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-18<br />
|SpyCatcher-Receptor:nLuc-Spytag (Catcher:nLuc-Tag)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159213 BBa_K1159213]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-19<br />
|SpyTag-Receptor:SpyCatcher-nLuc (Tag:Catcher-nLuc)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159214 BBa_K1159214]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-20<br />
|SpyTag-Receptor:nLuc-SpyCatcher (Tag:nLuc-Catcher)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159215 BBa_K1159215]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-21<br />
|Alcohol acetyltransferase I (Banana)<br />
|[http://parts.igem.org/Part:BBa_J45014 BBa_J45014]<br />
|<html><img src="https://static.igem.org/mediawiki/2013/0/09/TUM13_checkmark.png" width="25px" class="checkmark"></img></html><br />
|-<br />
|PF-22<br />
|Kill-switch with PIF3 (PIF-3)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159107 BBa_K1159107]<br />
|no<br />
|-<br />
|PF-23<br />
|Kill-switch with PIF6 (PIF-6)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159108 BBa_K1159108]<br />
| no<br />
|-<br />
|PF-24<br />
|Kill-switch with PIF6 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159120 BBa_K1159120]<br />
| no<br />
|-<br />
|PF-25<br />
|Kill-switch with PIF3 (FRET)<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159119 BBa_K1159119]<br />
| no<br />
|-<br />
|}<br />
<br />
===2. Preparation of linear DNA===<br />
In order to transfrom the moss, the DNA including our BioBricks with the nptII selection casette had to be linearized. We used EcoRI for the preparative digestion of our midipreps and simultaneously ran an analytical digest as quality control. Our preparations continued by purifying our digested DNA through salt, isopropanol and ethanol precipitation, solubilising in a small amount of water, measuring the concentration at the NanoDrop and suspending it in different volumes of sterile filtered Ca(NO<sub>3</sub>)<sub>2</sub> buffer for the intended concentration of 0.25 µg/µl .<br />
[[File:TUM13_Midiprep_picture1.png|thumb|left|450px|'''Figure 5''': Louise and Christopher preparing midipreps]]<br />
[[File:TUM13 Midiprep picture2.png|thumb|right|420px|'''Figure 6''': Midipreps for the moss transformation]]<br />
[[File:TUM13_Midiprepgel.png|thumb|center|910px|'''Figure 7''': Analytical gelelectrophoresis of linearized DNA constructs]]<br />
<br />
===3. Transformation of ''Physcomitrella patens''===<br />
To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol. <br />
<br />
The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium. <br />
<br />
The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.<br />
<br />
====Trips to Freiburg ====<br />
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We had the great chance to perform our '' '''Physcomitrella patens''' '' transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us. <br />
<br />
We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around. <br />
<br />
Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!<br />
<br />
===4. Regeneration and Selection of transgenic plants===<br />
After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.<br />
<br />
== Testing cultivation conditions ==<br />
===Growing ''Physcomitrella patens'' on solid materials===<br />
<br />
For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a [https://2013.igem.org/Team:TU-Munich/Results/Implementation#Our_swimming_remediation_raft remediation raft] since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.<br />
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<br />
===Determination of growth rates for different liquid culture forms===<br />
In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.<br />
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<li><img src="https://static.igem.org/mediawiki/2013/thumb/4/4a/TUM13_Tube_reactor_Ingmar.png/350px-TUM13_Tube_reactor_Ingmar.png" title="Tube reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/8/85/TUM13_Plate_reactor.png/350px-TUM13_Plate_reactor.png" title="Plate reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/7/77/TUM13_wave_reactor1.png/350px-TUM13_wave_reactor1.png" title="Wave reactor" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/thumb/b/ba/TUM13_wave_reactor2.png/350px-TUM13_wave_reactor2.png" title="Wave reactor" /></li><br />
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In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).<br />
<br />
[[File:TUM_13__growth_conditions.png|thumb|left|340px| '''Figure 1''': Different experimental setups for the growth of wild type moss in liquid culture. '''A:''' Aerated flasks, '''B:''' Standing flask, '''C:''' Shaken flask]]<br />
[[File:TUM13_growth_liquid_culture.png|thumb|right|520px| '''Figure 2:''' Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.]]<br />
All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. <br />
Normally at the [https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Reski laboratory] the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.<br />
<br />
===Tolerance to relevant environmental pollutants and toxins===<br />
<br />
To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2). <br />
<br />
[[File:TUM13_toxassay.png|thumb|center|900px| '''Figure 3:''' Toxicity assay for wild type moss]]<br />
<div class="visualClear"></div><br />
<br />
{|<br />
|+ '''Table 1''': Typical concentrations of toxic substances <br />
! Substance<br />
! Application<br />
! Concentration<br />
|-<br />
| Ampicillin<br />
| Antibiotic agar plate<br />
| 0.1 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Chloramphenicol<br />
| Antibiotic agar plate<br />
| 0.025 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Kanamycin<br />
| Antibiotic agar plate<br />
| 0.05 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Tetracycline<br />
| Antibiotic agar plate<br />
| 0.01 g/L<sup>[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf<nowiki>[1]</nowiki>]</sup><br />
|-<br />
| Geneticin (G418)<br />
| Antibiotic agar plate (for moss selection)<br />
| 12.5 mg/L<sup>[http://www.plant-biotech.net<nowiki>[2]</nowiki>]</sup><br />
|-<br />
|-<br />
| Diclofenac<br />
| 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) <br />
| 4.2-8.3 mg/L<br />
|-<br />
| NaCl<br />
| Sea water<br />
| 3.5 g/L<br />
|-<br />
| Catechol<br />
| Death of Arabidopsis<br />
| 55 mg/L<sup>[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/<nowiki>[3]</nowiki>]</sup><br />
|-<br />
| Erythromycin<br />
| 1 tablet (500 mg) dissolved in 6 L (blood circuit)<br />
| 0.08 g/L<br />
|}<br />
<br />
The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended.<br />
Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1).<br />
Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.<br />
<br />
<br />
==References:==<br />
<br />
[[http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/Working%20concentrations%20and%20stock%20solutions.pdf University of Toledo, Department of Environmental Sciences]] Working concentrations of various antibiotics<br />
<br />
[[http://www.plant-biotech.net plant-biotech.net ]] Prof. Reski´s Homepage<br />
<br />
[[https://static.igem.org/mediawiki/2013/1/18/Physcomitrella_cell_culture_conditions.pdf Physcomitrella cell culture conditions]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1586047/ Liao,Y. et al, 2006]] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. ''Plant Physiology'', 142(2): 620–628<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/RecombinantTeam:TU-Munich/Results/Recombinant2013-10-29T03:19:16Z<p>FlorianA: </p>
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==Characterization of recombinant effector proteins==<br />
[[File:Bild_Effectors.png|thumb|left|160px| '''Figure 1:''' Effectors]]<br />
<br />
For the development of a transgenic water filter it is an essential task to create a collection of well described and functional effector proteins which are either able to bind ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) or to degrade ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) xenobiotics which are present in the aquatic environment. This task was completed by the production of relevant effector proteins in ''E. coli'' and their subsequent purification and characterization. Deliberately we have chosen some well established BioBricks from the last years such as a laccase (<partinfo>BBa_K1159002</partinfo>) or the catechol dioxigenase (<partinfo>BBa_K648011</partinfo>) to improve these BioBricks. Beside these improvements we also added new BioBricks to the registry which we characterized ''in vitro'' such as the erythromycin esterase (EreB) (<partinfo>BBa_K1159000</partinfo>) or the NanoLuc luciferase (<partinfo>BBa_K1159001</partinfo>) which will be a useful tool for subsequent generations of iGEM students. For technical questions on our experiments, please see [https://2013.igem.org/Team:TU-Munich/Notebook/Methods protein biochemical methods] for further information. Beside these experiments with recombinant proteins we also characterized our stable transformed moss strains ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss see PhyscoFilter section]).<br />
<br />
{|cellspacing="0" border="1" right<br />
|+ '''Table 1:''' Investigated Proteins<br />
!Protein<br />
!BioBrick<br />
!RFC<br />
!Size [kDa]<br />
!Disulfide bridges<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#Eryhtromycin_Esterase_.28EreB.29 <b>Erythromycin esterase</b>] <br />
|[http://parts.igem.org/Part:BBa_K1159000 BBa_K1159000]<br />
|RFC[25]<br />
| align=right | 48.5<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#Laccase <b>Laccase</b>]<br />
|[http://parts.igem.org/Part:BBa_K1159002 BBa_K1159002]<br />
|RFC[25]<br />
| align=right | 58.8<br />
|yes<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#Nano_Luciferase <b>Nano Luciferase</b>]<br />
|[http://parts.igem.org/Part:BBa_K1159001 BBa_K1159001]<br />
|RFC[25]<br />
| align=right | 19.4<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#Catechol_Dioxigenase_.28XylE.29 <b>XylE</b>]<br />
|[http://parts.igem.org/Part:BBa_K648011 BBa_K648011]<br />
|RFC[10]<br />
| align=right | 4 x 35.4<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#Protein_phosphatase_1_.28from_Homo_sapiens.29 <b>PP1</b>]<br />
|[http://parts.igem.org/Part:BBa_K1159004 BBa_K1159004]<br />
|RFC[25]<br />
| align=right | 37.4<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#DDT-Dehydrochlorinase <b>DTT Dehydrochlorinase</b>]<br />
|[http://parts.igem.org/Part:BBa_K620000?title=Part:BBa_K620000 BBa_K620000]<br />
|RFC[10]<br />
| align=right | 23.4<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#SpyCatcher_.26_SpyTag <b>SpyCatcher</b>]<br />
|[http://parts.igem.org/Part:BBa_K1159200 BBa_K1159200]<br />
|RFC[25]<br />
| align=right | 12.6<br />
|none<br />
|-<br />
||[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#SpyCatcher_.26_SpyTag <b>SpyTag</b>]<br />
<br />
|[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159201 BBa_K1159201]<br />
|RFC[25]<br />
| align=right | 1.9<br />
|none<br />
|-<br />
|[https://2013.igem.org/Team:TU-Munich/Results/Recombinant#YFP_TEV_CFP <b>YFP_TEV_CFP</b>]<br />
|[http://parts.igem.org/Part:BBa_K1159112 BBa_K1159112]<br />
|RFC[25]<br />
| align=right | 55.2<br />
|none<br />
|}<br />
<br />
==Erythromycin Esterase (EreB)==<br />
The erythromycin esterase (EreB) is an enzyme found in some strains of ''Escherichia coli'' and which was introduced to the Parts Registry by the TU Munich iGEM Team 2013 in RFC[25]. As this BioBrick is derived from ''Escherichia coli'' it was to expect that it is well expressing in our experiments on recombinant effector proteins. For further information on the theoretical background of EreB please see our [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation page].<br />
<br />
===Production and purification of recombinant EreB===<br />
[[File:TUM13_P917.png|thumb|left|320px| '''Figure A:''' Streptavidin affinity chromatography for the erythromycin esterase]]<br />
[[File:TUM13_Analytprep_EreB.png|thumb|right|300px| '''Figure B:''' Analytical size exclusion chromatography on a Superdex 200 10/30 column]]<br />
[[File:TUM13_Preparative_EreB.Png|thumb|right|300px| '''Figure C:''' Preparative size exclusion chromatography on a Superdex 75 16/60 column]]<br />
[[File:TUM13_SDS_EreB.png|thumb|right|300px| '''Figure D''': SDS-gel of recombinant EreB with the marker (M) followed by the concentrated throughput of the streptavidin affinity column and 6 fractions collected from the elution peak]]<br />
The recombinant production and purification was carried out twice, in a first attempt 2 L of LB-media were used for an analytical purpose whereas in the second attempt we produced enough purified enzyme for all subsequent experiments. This preparation was carried out in 6 x 2L of LB media. Protein production was in both cases induced at OD = 0.8 by adjusting the cell culture to 5 mM of arabinose and was carried out for 4 h for the first and 5 h for the second preparation. Cell disruption was performed by ultrasonic sound in both cases. The cell lysate was then dialyzed against 5 L of SA-buffer and subsequently applied to streptavidin affinity columns. After the application of the protein, the column was washed with SA-buffer until a base line was reached. Afterwards the protein was eluted using 5 mM biotin. During the first preparation 2-mercapto-ethanol was added after the chromatographic steps. In order to avoid oxidation of cysteine residues to disulphid-bridges, which is not desired for the cytosolic EreB protein, the preparative purification was carried out with buffers containing 5 mM of 2-mercapto-ethanol in all buffers. When comparing the size exclusion chromatograms, obtained from the analytical and the preparative purification, it can be stated that there is still a considerable aggregation peak near the void volume (Fig. B) of the column in the first attempt, which was nearly not the case for the preparative preparation (Fig. C). Therefore we would give the advise to use strictly reducing conditions while working with recombinant EreB. The finally resulting yields of the preparative purification have been determined by absorption measurement of the aromatic amino acids at 280. The total yield was determined to 25 mg of pure protein which is 2.1 mg/L of LB culture.<br />
<br />
===Kirby-Bauer Assay: Measuring remaining erythromycin on a pertri dish===<br />
[[File:TUM13_Kirby_Bauer.png|thumb|left|400px| '''Figure 2:''' Kirby-Bauer assay of recombinant EreB incubated with erythromycin]]<br />
The Kirby-Bauer assay is an '''agar diffusion test''', with which it is very easy to examine the decomposition activity of the enzyme. We also analyzed the decomposition activity of the recombinant EreB by [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin LC-MS]. The recombinant protein is incubated with the antibiotic and the reaction is stopped with methanol. To be sure that the enzyme is inhibited and the reaction does not go on, we additionally heated the reaction mixture (see table 2) after stopping for two minutes at 50°C and then shock froze the mixture in liquid nitrogen. The effect of methanol, heating and shock freezing on the bacterial strain and the substrate were checked before and have no influence. <br><br />
<br />
Then bacteria from a quite dense liquid culture are plated on a LB agar plate without antibiotics under sterile conditions and spread with sterile cotton tip applicators. We used the bacterial strain ''Micrococcus luteus'' which was generously donated from the [http://www.micbio.wzw.tum.de/cms/docs/scherer-anzeigen.php chair of microbiology by Prof. Scherer`s group] and is mentioned to be specifically sensitive to Erythromycin [[http://pubs.acs.org/doi/abs/10.1021/bi201790u?mi=0&af=R&pr... Wright et al., 2012]]. Now several 6 mm filter paper discs are placed on the bacterial lawn in adequate intervals and 8 µl of the spinned down reaction mixture is added onto one disc. The mixture diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease with increasing distance. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is called the '''zone of inhibition'''.<br />
<br />
{|<br />
|+ '''Table 2:''' Reaction mixture for Kirby-Bauer assay<br />
! substance<br />
! amount<br />
! stock solution<br />
|-<br />
| EreB recombinant protein 40 nM<br />
| align=right | 0.18 µl<br />
| 11 µM EreB in PBS with 10 mM ß-mercaptoethanol, 2% glycerol (v/v) and 300 mM NaCl<br />
|-<br />
| Erythromycin 0.36 mM<br />
| align=right | 3 µl<br />
| Erythromycin in ethanol, 6 mM<br />
|-<br />
| Tris-HCl buffer pH 7.5 100 mM<br />
| align=right | 10 µl<br />
| Tris-HCl buffer pH 7.5, 500 mM<br />
|-<br />
| NaCl 0.08 M<br />
| align=right | 4 µl<br />
| NaCl in water, 1 M<br />
|-<br />
| ddH20<br />
| align=right | 32.82 µl<br />
| ddH20<br />
|-<br />
| '''TOTAL:'''<br />
| align=right | '''50 µl'''<br />
|<br />
|}<br />
<br />
As negative controls there is one mixture containing no substrate or enzyme and two mixtures without the enzyme but with antibiotic, which were incubated for 0 minutes and 6 hours. <br />
<br />
As expected the assay shows a gradually decreasing zone of inhibtion with increasing reaction time. In the first half hour the zone is more or less constant although the enzyme is constantly degrading the antibiotic. This can be explained by the fact that the mixture only diffuses a certain range into the agar and in the first few time steps of the reaction the concentration of the antibiotic within this range is above the [http://en.wikipedia.org/wiki/Minimum_inhibitory_concentration minimum inhibitory concentration (MIC)], so the bacteria is inhibited independently of the exact concentration. After 3 hours all Erythromycin was degraded so there is no zone of inhibition anymore. Also we can see that the antibiotic does not degrade by itself significantly over 6 hours since the zones of inhibition in both negative controls are pretty much the same.<br />
<br />
===Characterisation of the enzyme===<br />
[[File:TUM13_PNBT_reaction.png|thumb|right|450px| '''Figure 3:''' Reaction mechanism of EreB hydrolyzing the model ester p-NPB into a chromogenic product]]<br />
To characterize the enzyme we performed a spectrophotometric assay using the model ester p-nitrophenyl-butyrate (p-NPB) as a substrate for EreB. The chromogenic product p-Nitrophenyl shows characteristic absorbance at 405 nm. The reaction takes place even faster than hydrolysis of erythromycin since p-NPB is already highly activated and therefore readily hydrolyzed. This is also the reason why it degrades quite rapidly by itself so reactions have to be measured soon after being set up and taking a negative control is especially important.<br />
<br />
====Kinetics====<br />
<br />
[[File:TUM13_EreB_plate1.jpeg|thumb|right|300px| '''Figure 4:''' 96-well plate showing the substrate serial dilution incubated with EreB (1-4) and as negative control with water (0)]]<br />
To determine the kinetic parameters of EreB we prepared a 100 µl serial dilution of the substrate, starting with 1.2 mM p-NPB (in 0.4% Triton, 50 mM buffer Tris-HCl pH 7.5, 0.2 M NaCl) to which 100 µl of the enzyme (11 µM) were added. The experiment was performed in quadruplets and the mean value was then plotted (see figure 5) against substrate concentrations.<br />
<br />
[[File:TUM13_EreB_kinetic.jpeg|thumb|right|900px| '''Figure 5:''' Reaction of EreB with a serial dilution of p-NPB (left) and negative control without the enzyme (right)]]<br />
<br />
'''Result''': The higher the substrate concentration the faster the substrate is hydrolyzed, which can be seen in an increasing gradient and is self-evident as the enzyme encounters the substrate more often and faster the higher its concentration is in the solution. This data was also used for an exact determination of the kinetic parameters in [https://2013.igem.org/Team:TU-Munich/Modeling/Enzyme#Erythromycinesterase_.28Substrate_Dependence.29 our modelling section]. At these reaction conditions (pH 7.5) almost no decomposition of p-NPB takes place which is confirmed in the following experiment.<br />
<br />
====PH optimum====<br />
<br />
<br />
We prepared a buffer series from pH 4.5 to pH 10 using Sodium acetate + acetic acid (pH 4, 4.5, 5), MES (pH 5.5, 6), HEPES (6.5, 7), Tris-HCl (7.5, 8, 8.5) and Ches (9, 9.5, 10), each 100 µl in a 96-well plate. To the buffer we added 50 µl Substrate p-NPB (2 mM in 0.8% Triton and 400 mM NaCl) and finally 100 µl of the enzyme (11 µM) to start the reaction. The reaction was followed with a spectrophotometer at 405 nm for 1 hour every 15 seconds with the reaction mixture being shaken before every read. The experiment was performed threefold and the mean value was then plotted (see figure 6) against the pH-value of the buffer.<br />
<br />
[[File:TUM13_EreB1.png|thumb|left|900px| '''Figure 6:''' Reaction of EreB with p-NPB in different pH buffers (left) and negative control without the enzyme (right)]]<br />
<br />
'''Result''': From pH 4.5 to 6.5 there was no enzyme activity because the enzyme degraded, which was clearly visible as a dreary solution. The small absorption can be traced back to the degraded enzyme, so the conclusion is that EreB is unstable in alcaline surroundings. This is also the reason why [https://2013.igem.org/Team:TU-Munich/Modeling/Enzyme#Erythromycinesterase_.28Substrate_Dependence.29only cytoplasmatic localized enzyme works in our transformed moss], because the Knop medium in which ''P. patens'' is grown has a pH of 5.8. Since natural aquatic environment is generally on the alcalic side the degradation of antibiotics with EreB can only be realized intracellular. P-NPB in contrast seems to be very stable at low pH as there is no absorption measured in the negative control. With increasing pH the substrate increasingly degrades by itself while the enzyme's activity is also increasing. These two factors lead to a rapidly growing conversion of p-NPB. In a nutshell EreB works optimally in an acidic environment.<br />
<br />
====Ionic strength optimum====<br />
<br />
A serial dilution of NaCl starting with 1.5 M was prepared (50 µl) and another 100 µl of p-NPB (2 mM in 0.8 % Triton) were added before the reaction was started with 50 µl of the enzyme (11 µM). The reaction was followed with a spectrophotometer at 405 nm for 1 hour every 20 seconds with the reaction mixture being shaken before every read. The experiment was performed threefold and the mean value was then plotted (see figure 7) against the varying salt concentrations.<br />
<br />
[[File:TUM13_EreB2.png|thumb|left|900px| '''Figure 7:''' Reaction of EreB with p-NPB at different salt concentrations and negative control without the enzyme (right)]]<br />
<br />
'''Result''': The lower the salt concentration the higher is the enzymatic activity while simultaneously the decomposition of p-NPB increases. However, the reaction does not reach saturation as in the reaction with different pH (see figure 6). This is intuitive since the double amount of substrate and half of the enzyme's amount were incubated.<br />
<br />
==Laccase==<br />
[[File:TUM13 Animation_Laccasex.gif|thumb|right|320px| '''Figure 8:''' Animation of protein structure of Laccase]]<br />
Laccases are an important group of effector proteins that can be utilized in bioremediation to degrade xenobiotics. They are copper-containing enzymes which catalyze the oxidation of a wide range of substances. It was interesting for us as the two important xenobiotics ethinylestradiol and diclofenac can be degraded using laccases and the general function of the enzyme can easily be investigated using the commonly used substrate ABTS which results in a chromogenic product.<br><br />
During the planing phase of our project we decided not to add a new laccase to the Parts Registry but instead to continue the work on the best availible laccase. The iGEM team [https://2012.igem.org/Team:Bielefeld-Germany Bielefeld Germany] 2012 has created BioBricks for five different laccases during the last summer. So we contacted this iGEM team and got the advise that the laccase derived from ''Bacillus pumilus'' <partinfo>BBa_K863000</partinfo> seems to be the best choice for our effector collection. As we wanted to try this effector protein in different localisations such as cytoplasmatic, secreted, or receptor bound we converted this enzyme to RFC[25] in order to make it compatible for protein fusions.<br />
<br />
===Bioinformatics: Laccase - a secreted enzyme===<br />
[[File:TUM13_Laccase_DSB.png|thumb|right|320px| '''Figure 9:''' Structural analysis of Laccase by homology]]<br />
For the production of effector proteins it is always essential to know whether they are naturally cytoplasmatic or whether they are secreted. Cytoplasmatic proteins usually contain a higher portion of cysteine residues as no disulfide bridges can be formed under the reducing conditions of the cytoplasm. In contrast secreted proteins often do contain disulfide briges which make them more stable to environmental stress. The disulfide formation is possible for the secreted proteins as the oxidizing extracellular conditions facilitate the bridge formation. On the other hand there is a selection pressure for secreted proteins not to contain exposed cysteine residues that are not involved in the formation of disulfide bridges. Such free cysteine residues would lead to aggregation of proteins which would probably hamper their activity. Therefore secreted enzymes generally only contain a smaller amount of cysteine residues. <br><br />
Thus it is important to find out whether the used laccase BPUL is a secreted enzyme as this would increase the chance to express it in a functional form on the extracellular part of our moss. To solve this question we did an alignment of the laccase BioBrick and the closest related solved structure. The structure contains disulfide bridges for example Cys<sup>229</sup>-Cys<sup>322</sup>. The comparison of the position of these residues within the amino acid sequence alignment shows that these residues are conserved and it can thus be assumed that the laccase BioBrick we used also contains disulfide bridges that have to be formed under oxidizing conditions and will increase the stability of our effector protein.<br><br />
We thus used 10 mM of potassium ferrocyanide which is a milde oxidization reagent subsequent to the cell disruption and did not use any 2-mercaptoethanol as a reducing agent during the protein purification.<br />
<br />
===Analytical preparation===<br />
[[File:TUM13_Laccase_Chromatogramm.png|thumb|left|350px| '''Figure 10:''' Streptavidin affinity chromatography]]<br />
[[File:TUM13_Preparative_Laccase.png|thumb|right|320px| '''Figure 11a:''' Size exclusion chromatography on a Superdex 200 10/30 column]]<br />
[[File:TUM13_SDS_laccase.png|thumb|right|320px| '''Figure 11b:''' SDS-gel of recombinant Laccase with the marker (M) followed by the the cell lysate, concentrated protein throughput of the streptavidin affinity column, 1 fraction of the eluate unconcentrated and concentrated]]<br />
As with all other effector proteins we performed at first an analytical preparation in a 2 L LB-media scale in order to learn about the protein and then up-scaled the production to 6 x 2 L of LB media. The production was carried out cytoplasmatically in ''E. coli'' BL21. The culture was grown to OD 0.8 and then induced using 5 mM of arabinose and the protein production was carried out for 5 hours. Cell disruption was performed using ultrasonic sound and subsequently the cell lysate was dialyzed against 1 x SA-Buffer that contained additional 5 mM of the milde oxidation reagent potassium ferrocyanide in order to form the disulfide bridges present in the laccase structure (see previous paragraph).<br><br />
The streptavidin affinity chromatography yielded a satisfying elution peak that was then concentrated using a centrifugal filter unit (MWCO: 30 kDa). The concentrated protein was then applied to an ÄKTA purifier equipped with a Superdex 200 10/30 size exclusion chromatography column. The chromatogram shows three major peaks with a first peak near the void volume that corresponds to aggregated protein >600 kDa, a second elution peak of high molecular protein and a third elution peak corresponding to the desired laccase protein. This assumption was made on the knowledge that on this column proteins of arround 50 kDa elute at an elution volume of 14 to 15 ml. This assumption was subsequently confirmed by ABTS activity assays with all elution fractions (see next section).<br />
<br />
===Activity determination using ABTS===<br />
[[File:TUM13_ABTS-oxidation.png|thumb|left|250px| '''Figure 12:''' Mechanism of the oxidation of ABTS which is a model substrate for laccase activity]]<br />
[[File:TUM13_Laccase_activity.png|thumb|right|320px| '''Figure 13:''' Correlation of size exclusion chromatography (blue) and activity determination using ABTS (red). Relative values are shown.]]<br />
The enzymatic activity of the purified laccase was determined by the ABTS-assay. In a first pre experiment the appropriate dilution factor was determined to be 100-fold. The elution fractions obtained from size exclusion chromatography were diluted 1:100 in PBS and in an ELISA plate. 100 µl of the enzyme and 100 µl of ABTS substrate were mixed and a kinetic measurement at 405 nm was performed. The absorption at 280 nm in the SEC chromatogramm (blue) identifies three main protein peaks, with a first peak corresponding to aggregated protein, a shoulder which also corresponds to higher molecular protein and a single peak which was proposed to be the monomeric laccase. The relative activity obtained for the different elution fractions was plotted in the same diagramm and shows a clear peak which matches the laccase peak in the SEC. Beside this major peak a second smaller peak of active fraction was visible which appeared in earlier elution fractions and might correspond to dimerized laccase. As the laccase is a secreted enzyme which also bears dislufide bridges it was produced in the cytoplasm and subsequently it was oxidized to form the proper disulfide bonds. As this process might be only partial there is a possiblity for the formation of disulfide dimers. Nevertheless the fractions 14 to 17 were pooled for further experiments as they showed the highest enzymatic activity. The protein concentration of the pooled fraction was determined to be 0.48 mg/ml after SEC.<br />
<br />
===Characterization of the enzyme===<br />
The enzymatic activity of the laccase BioBrick was investigated under different conditions as this was important for our [https://2013.igem.org/Team:TU-Munich/Modeling/Filter filter model] in which we try to simulate the application of the laccase in our remediation raft to clean highly contaminated rivers. The first activity assays were dilution series experiments in which we wanted to determine the appropriate amount of protein which we had to apply in the assays. We found that a 1:100 dilution of the prepared protein (0.49 mg/ml) to be ideal and thus performed all experiments with this concentration. <br />
====Kinetic assay (Substrate dependency)====<br />
[[File:TUM13 Laccase_plate1.jpeg|thumb|left|400px| '''Figure 14:''' 96-well plate showing dilutin series of substrate (ABTS) for kinetic experiments]] For the kinetic assay a 1:1 dilution series of the substrate was set up. The oxidation of the ABTS substrate was then monitored over time observing the absorbance at 405 nm. The experiment was performed in triplicates (see Figure 14) and as a control the dilution series of the substrate was incubated with PBS instead of the diluted protein. It can be seen that there is no autolytic oxidation of the substrate. The resulting kinetic parameters are summarized in table 3.<br />
<br />
{|<br />
|+ '''Table 3:''' Best fit parameters<br />
! Parameter<br />
! Best fit<br />
|-<br />
| k<sub>f</sub><br />
| 1.606 E+05 1/s<br />
|-<br />
| k<sub>r</sub><br />
| 3.450 E-08 1/s<br />
|-<br />
| k<sub>cat</sub><br />
| 63.522 1/s<br />
|-<br />
| K<sub>M</sub><br />
| 0.396 mM<br />
|}<br />
<br />
For a detailed evaluation of the results please see our [https://2013.igem.org/Team:TU-Munich/Modeling/Enzyme#Laccase_.28Substrate_Dependence.29 enzyme kinetics] modeling.<br />
[[File:TUM13 Laccase_kinetic.png|thumb|left|900px| '''Figure 15:''' Reaction of Laccase with a serial dilution of ABTS (left) and negative control without the enzyme (right)]]<br />
<br />
====Activity dependency on pH values====<br />
Secondly we did a series of experiments in which we determined the effect of the pH on the enzymatic activity (see Figure 16). For these experiments 100 µl of various 500 mM buffer (various pH), 50 µl of ABTS substrate (0.5 mg/ ml in ABTS-bufer from Roche) and 50 µl of laccase, diluted 1:100, were incubated. The absorbance at 405 nm was then recorded over the time in a 96-well plate. <br />
The change in absorbance over time is shown for every condition as the average of three measurements with standard deviation. The resulting plot shows that there is an optimum for the pH which is around 5. This matches the acidic conditions present in the apoplast of the plant, which is mainly between pH 5 and pH 6.5, so localizing the enzyme in the interspace between cell wall and membrane is theoretically possible. <br />
<br />
[[File:TUM13 Laccase_pH.png|thumb|left|900px| '''Figure 16:''' Reaction of Laccase with ABTS in different pH buffers. Absorbance curve with laccase on the left; control with PBS on the right]]<br />
<br />
====Influence of ionic strength====<br />
Beside the substrate dependence and the influence of the pH value, the dependence of the Laccase on the ionic strength of the media was of interest for our project as well as is is for further users of this BioBrick. We thus incubated the recombinant laccase (1:100 dilution) with ABTS substrate and various concentrations of sodium chloride. This was done in triplicates and for a control without the enzyme with PBS instead.<br />
[[File:TUM13 Laccase_Salz.png|thumb|left|900px| '''Figure 17:''' Reaction of laccase with ABTS at different salt concentrations. Absorbance curve with laccase on the left; control with PBS on the right]]<br />
As it can clearly be seen the enzymatic activity is increasing with decreasing salt concentration. For a detailed analysis of the results please look at our [https://2013.igem.org/Team:TU-Munich/Modeling/Enzyme enzyme modelling page].<br />
<br />
====Influence of the temperature====<br />
<br />
[[File:TUM13 Laccase_Temperature.png|thumb|left|400px| '''Figure 18:''' Reaction of laccase with ABTS at different temperatures]]<br />
<br />
To analyze the temperature dependency of the laccase we determined the katalytic activity with a 1:100 enzyme dilution and 0.12 mM of the substrate ABTS. The enzymatic conversion could be followed by the absorbance of the resulting product at 405 nm. The enzyme and substrate were both incubated at the specific temperatures for half an hour before starting the reaction. <br />
As expected the enzymes efficiency is increasing with temperature, but it is also acceptable at lake and river temperatures which are about 10 to 15 °C. <br />
<div class="visualClear"></div><br />
[[File:TUM13 Laccase_TempAuswertung.png|thumb|left|400px| '''Figure 19:''' Dependence of the activity of the laccase on the ambient temperature]]<br />
<br />
For our [https://2013.igem.org/Team:TU-Munich/Modeling/Filter filter modelling] we determined k<sub>cat</sub>/K<sub>M</sub> from these measurements by looking at the initial gradient. We normalized this by equating the enzyme activity at 20° C with the activity determined by our [https://2013.igem.org/Team:TU-Munich/Modeling/Enzyme modeling] of the substrate dependence of the laccase. This can be assumed, because in the experimental setup the substrate concentration is a lot less than the K<sub>M</sub> value we determined, which then simplifies the Michaelis-Menten equation to give, that the initial gradient is proportional to the enzyme activity k<sub>cat</sub>/K<sub>M</sub>.This gave us the activity dependence shown in Figure 19.<br />
<br />
====Half-life of the laccase in river water====<br />
<br />
[[File:TUM13 Laccase_0std.png|thumb|left|400px| '''Figure 20:''' Laccase reaction after 0 hours of incubation in river water]]<br />
<br />
Laccase with an initial concentration of 0,3125 µM was diluted with 1:1 steps at the initial time, then ABTS at a concentration of 97.2 µM was added to each dilution and the kinetic of the ABTS reaction was recorded using a photometer at 405 nm. Figure 20 shows the results.<br />
<br />
Then the laccase was dialyzed against river water, taken from the local river Isar. After 96 hours and again after 144 hours, the initial dilution experiment was repeated giving figure 21 any 22, respectively. It is apparent that the enzymes activity has been reduced. <br />
From these measurements we '''estimated the half-life to be 36.3 hours''', corresponding to an inactivation rate of 5.3 10<sup>-6</sup> s<sup>-1</sup>, which we then used in our [https://2013.igem.org/Team:TU-Munich/Modeling/Filter filter model].<br />
<br />
[[File:TUM13 Laccase_96std.png|thumb|left|400px| '''Figure 21:''' Laccase reaction after 96 hours of incubation in river water]]<br />
[[File:TUM13 Laccase_144std.png|thumb|right|400px| '''Figure 22:''' Laccase reaction after 144 hours of incubation in river water]]<br />
<br />
==Nano Luciferase==<br />
The Nano Luciferase (NanoLuc) which was introduced in 2013 by Promega is a new member of the luciferase reporter gene/protein familiy and shows some advantages compared to the other family members. The NanoLuc is very small (19 kDa) compared to the firefly luciferase (61 kDa) and the ''Renilla'' luciferase (36 kDa). On the other hand it is also said that the specific activity of the NanoLuc is about 150-fold stronger compared to conventional luciferases and the background caused by autoluminescense of the substrate is said to be remarkably smaller. <br />
<br />
===Production in ''E. coli'' and purification===<br />
[[File:TUM13_Analytprep_NanoLuc.png|thumb|right|320px| '''Figure 23:''' Analytical size exclusion chromatography on a Superdex 200 10/30 column showing a single elution peak for the NanoLuc.]]<br />
[[File:TUM13_Preparative_NanoLuc.png|thumb|right|320px| '''Figure 24:''' Preperative size exclusion chromatography on a Superdex 75 10/30 column showing a single elution peak for the NanoLuc.]]<br />
[[File:TUM13_Nanoluciferase_chromatogramm.png|thumb|left|320px| '''Figure 25: '''Streptavidin affinity chromatography for NanoLuc]]<br />
Therefore the NanoLuc was synthesized as a BioBrick in RFC[25] and was produced in ''E. coli'' using the pBad expression system with a C-terminal ''Strep''-tag. After the production (2 l of LB-media for analytical and 12 l for preparative preparations) the cells were disrupted using sonification and the lysate was dialysed against 5 l of 1x SA-buffer. Afterwards the lysate was applied to a Streptavidin-Affinity (SA) column and was subsequently washed using SA-Buffer until a baseline was reached and the protein was then eluted using 5 mM of biotin (Attention: These are special columns which are not availible commercially. If you are using commercial colum material you have to use d-Desthiobiotin because usual biotin will elute your protein but you will not be able to regenerate the column after your chromatography). After the SA-chromatography the protein was concentrated using centrifugal concentration units (MWCO: 10 kDa). The concentrated protein was then applied onto a Superdex S200/75 size exclusion chromatography. The chromatogram of both preparations show a single peak in the chromatogram which elutes at an expected elution volume of 15 ml. The absence of any notable aggregation peak shows the high stability of this protein and the ease of production.<br />
<br />
[[File:TUM13_SDS_nLuc.png|thumb|right|320px|'''Figure 26:'''SDS-gel of recombinant nLuc with the marker (M) followed by the concentrated throughput of the streptavidin affinity column and 6 fractions collected from the elution peak]]<br />
<br />
===Structure of the Nano Luciferase===<br />
[[File:TUM13_CD_nLuc.png|thumb|right|300px| '''Figure 27:''' Circular dichroism spectrum of the recombinant produced NanoLuc luciferase]]<br />
[[File:TUM13_Annimated_test.gif|thumb|left|300px| '''Figure 28:''' Homologous structure (3ppt_A) of the NanoLuc luciferase]]<br />
There is no structure available for the [http://parts.igem.org/Part:BBa_K1159001 NanoLuc Luciferase] in the [http://www.rcsb.org/pdb/home/home.do Protein Data Bank]. In our [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions protein modelling] we used homolgy search and identified the structure [http://www.rcsb.org/pdb/explore/explore.do?structureId=3PPT 3ppt_A] as the solved structure with the highest homology to the NanoLuc which has only 21% identity with a similarity of only 0.359. The result of the homology search is shown as annimated gif in Figure 28 (please see our [https://2013.igem.org/Team:TU-Munich/Results/How_To How To] for an introduction). The protein was dialysed against 1x CD-buffer and subsequently a circular dichroism spectroscopy was taken (learn about [https://2013.igem.org/Team:TU-Munich/Notebook/Methods#Circular_Dichroism_Spectroscopy CD spectroscopy]). The CD spectrum was used to predict the secondary structure content of the NanoLuc which could be determined to 35.1% helix, 27.6% b-strand, 18.5% turn and 18.8% random. As there is only a poor homology present, a detailed comparison of the determined and the predicted secondary structure is not possible. But it can be stated that both show a balanced content of different secondary structures and that the produced protein is present in a folded conformation. The mixed secondary structure content is also in consistance with the predicted secondary structure shown in the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] sequence window ([http://parts.igem.org/Part:BBa_K1159001 click here]).<br />
<br />
===Activity determination of Luminescense===<br />
[[File:TUM13_nLuc_activity_pH.png|thumb|right|320px| '''Figure 29:''' Activity dependence of the NanoLuc luciferase on the pH.]]<br />
[[File:TUM13_nLuc_activity_NaCl.png|thumb|right|320px| '''Figure 30:''' Activity dependence of the NanoLuc luciferase on the ionic strength.]]<br />
The activity of the produced NanoLuc was investigated by its luminescense. The assay was performed in white 96-well plates (Nund) and the bioluminescense was determined at 460 nm in a BioTeK II plate reader for 1 sec per well. For every reaction 50 µl of the NanoGlow substrate (sponsored by Promega) was mixed with 50 µl of the NanoLuc preparation. In a first experiment a dilution series of the NanoLuc was performed in order to find the apropriate dilution of the enzyme (data not shown). Finally the 1:100 000 dilution was used for all experiments as it resulted in intermediate luminescense values within the dynamic range of the plate reader. <br><br />
The dependency of the luminescense reaction on the pH value of the buffer was assayed by an experiment in which the NanoGlow substrate was diluted in water instead of the provided buffer and the pH was adapted by 100 µl of 500 mM buffers. The resulting luminescense is shown in Figure 23 and shows a clear optimum around pH 8.0 to 8.5. A luminescense value of more than 50% can be expected for pH values between 6 and 9 which is sufficient for most assays. <br><br />
For the dependency of the luminescence on the ionic strength 50 µl of diluted NanoLuc, 50 µl of NanoGlow substrate and 100 µl of sodium chloride dilution series were incubated and afterwards the luminescense was quantified in triplicates. The result is impressing and again speaks for the NanoLuc luciferase as an innovative reporter protein. The obtained luminescense is constant from 0 to 250 mM of NaCl and upon 1 M of NaCl at least 50% of the maximal luminescense can be expected.<br />
<br />
==Catechol Dioxigenase (XylE)==<br />
[[File:TUM13 Animation_XylEx.gif|thumb|right|320px| '''Figure 25:''' Animation of protein structure of XylE]]<br />
The catechol dioxigenase (<partinfo>BBa_K648011</partinfo>) is a BioBrick which was characterized by the [https://2010.igem.org/Team:Imperial_College_London Imperial College 2010] iGEM team. As we also want to use this BioBrick we produced it as a recombinant protein. Given the fact that previous teams did a great job in characterizing this proteins kinetic parameters we decided to focus on other aspects.<br />
Catecholdioxygenases are a wide variety of enzymes degrading catechol and various of its derivatives. The xylE gene we use is from ''Pseudomonas putida'' and has a ferrous ion ligand in each subunit. The enzyme has the structure of a homotetramer<br />
<br />
<br />
<br />
===Analytical preparation===<br />
[[File:TUM13_XylE_chromatogramm.png|thumb|left|320px| '''Figure 26:''' Streptavidin affinity chromatography for the catecholdioxygenase]]<br />
To investigate if our recombinant BL21 ''E.coli'' cells were producing the catecholdioxygenase we started with an analytical preparation in 2 l LB-medium. After we confirmed the succesful transformation of our cells we produced another 2 x 2 l flasks full of recombinant cells in order to have enough enzyme to work with. To induce protein production we used 5 mM of arabinose when we measured an OD of 0.8. We disrupted the cells by sonification and after dialysis in 1 x SA-buffer we performed streptavidin affinity chromatography.<br />
After elution of the desired protein in 1 x SA-buffer with 5 mM 2-mercaptoethanol and 10% glycerol we concentrated it using a centrifugal filter unit (MWCO: 30 kDa). The concentrated protein was then applied to a ÄKTA purifier equipped with a Superdex 75 16/60 size exclusion chromatography column. As seen in the figure below the protein was eluted without any aggregation products and was ready to use.<br />
[[File:TUM13_XylE_analy_SEC2.png|thumb|right|320px|'''Figure 27:''' Size exclusion chromatography for the catecholdioxygenase]]<br />
<br />
==Protein phosphatase 1 (from ''Homo sapiens'')==<br />
[[File:TUM13 Animation_PP1.gif|thumb|right|320px| '''Figure 28:''' Animation of protein structure of PP1]]<br />
The production of recombinant protein phosphatase 1 was part of our collaboration with the Dundee iGEM team 2013. They developed this BioBrick which naturally binds [http://de.wikipedia.org/wiki/Microcystine microcystine], an important environmental toxin. <br />
===Production and purification of recombinant PP1 protein===<br />
[[File:TUM13_P913.png|thumb|left|320px| '''Figure 29:''' Streptavidin affinity chromatography of protein phosphatase 1]]<br />
[[File:TUM13_PP1_analy_preparation.png|thumb|right|320px| '''Figure 30:''' Size exclusion chromatography of protein phosphatase 1 on Superdex 200 10/30 column]]<br />
[[File:TUM13_SDS_PP1.png|thumb|right|320px| '''Figure 31:''' SDS-gel of recombinant PP1 with the marker (M) followed by the the cell lysate, concentrated protein throughput of the streptavidin affinity column, 1 fraction of the eluate unconcentrated and concentrated]]<br />
We converted this BioBrick to RFC[25] and subsequently cloned it into the expression vector pBad_C-terminal_''Strep''. Beside the recombinant characterisation we also created a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss transgenic moss transformed with a receptor containing PP1] in its extracellular domain. The recombinant protein production was carried out in ''E. coli'' BL-21 which was grown in 2 L of LB-media. The protein production was induced at OD 0.8 by addition of arabinose to a concentration of 5 mM. The cells were harvested after 4 h and subsequently resuspended in 20 mL SA-buffer with 5 mM 2-mercapto ethanol. Cell disruption was performed using ultrasonic sound. The dialysed cell extracte was applied to a streptavidin affinity column which was washed until a base line was reached and was subsequently eluted using SA-buffer containing 5 mM of biotin and 5 mM of 2-mercapto ethanol. The elution peak recorded by the continuous measurement of the absorbance at 280 nm indicated a good yield of recombinant protein (Fig. 29). As a second purification step after the streptavidin affinity chromatography we concentrated the protein in centrifugal filter units in order to apply it to a size exclusion chromatography. During the concentration process there were clear signal for precipitated protein which appeared as white flakes in the concentration filter unit. This effect was by far the most drastic precipitation of recombinant that was detected during this iGEM project. Anyhow the concentrated protein was centrifuged for 5 min at 13 200 RPM to remove particles of precipitated protein and was then applied to an ÄKTA purified with a Superdex 200 10/30 column (Fig. 30). There were four different peaks present in the chromatogramm which most probably correspond to (1) a peak created by aggregated protein which runs in the voit volume of the size exclusion colum, (2) a diffuse peak which might correspond to multimeric PP1, (3) a sharp peak at the elution volume where the recombinant PP1 was expected and (4) a peak which is most probably caused by low molecular buffer substances. The general signal intensity obtained in the size exclusion was very low indicating low protein concentrations. The maximal peak obtained for PP1 had an absorbance of 50 milli Absorption Units whereas this was for example for the NanoLuc in the range of 1000 for a comparable experiment. The fact that this protein does not tolerate the concentration procedure gives an indication that it is fragile and tends to aggregation when stress is applied. Finally recombinant PP1 protein was prepared although the yield was very low compared with other effector proteins.<br />
<br />
===Possible reasons for the instability of PP1 ''in vitro''===<br />
The high aggregation and denaturation tendency of the protein phosphatase 1 which was observed in the analytical preparation motivated the search for a possible explanation. As a first hint the amino acid composition calculated by the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] was examined and it can be seen that 13 cysteine residues are present in the sequence of the protein phosphatase. This corresponds to 4% of the total number of amino acid residues comapred to 2.8% cystein residues in an average protein. This information taken together with the molecular function of the protein phosphatase 1 clearly shows that this BioBrick is a cytoplasmatic protein which fulfills its function in the reducing milieu of the cytoplasm and tends to aggregation when exposed to an oxidizing milieu such a the periplasm of ''E. coli''. <br><br />
Secondly the amino acid sequence produced by the AutoAnnotator was fed into the bioinformatic tool [http://web.expasy.org/cgi-bin/protparam/protparam ProtParam] which gives a stability prediction for proteins. The result was: "The instability index (II) is computed to be 42.99 - This classifies the protein as unstable." <br><br />
Taken both these indications into account it still seems a good idea to express the protein phosphatase 1 cytoplasmatically to bind microcystine but it is not advisable to think about a secreted version of PP1 as the protein seems to unstable for this application.<br />
<br />
==DDT-Dehydrochlorinase==<br />
[[File:TUM13 Animation_DDT.gif|thumb|right|320px| '''Figure 32:''' Animation of protein structure of DDT]]<br />
The DDT dehydrochlorinase (<partinfo>BBa_K620000</partinfo>) BioBrick was designed in RFC[10] by the [https://2011.igem.org/Team:Caltech Caltech 2011] iGEM team and as a localization of this effector outside the cytoplasm isn't advisable we didn't improve this BioBrick to RFC[25]. The DDT dehydrochlorinase is dependent on the continuous supply of glutathione which conjugates to xenobiotics in order to inactivate them. The disadvantage of an RFC[10] BioBrick is that it can not be fused to an affinity tag which can subsequently be used to purify the protein after its recombinant production. <br> <br />
Beside the experiments already performed in iGEM we found monochlorobimane to be an interesting assay for experiments in bioremeditation. This commercially availible substrate can be conjugated to glutathione. The conjugation of monochlorobimane can be followed by the fluorescense of the conjugation product (excitation: 380 nm / emission: 461 nm). We tryed this assay with transgenic plants but could not see results as the autofluorescense of the photosystem in ''Physcomitrella'' was too strong in order to see the emergence of the fluorescense caused by the conjugation product.<br />
<br />
==References:==<br />
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[[http://udel.edu/~gshriver/pdf/Pimenteletal1997.pdf Pmentel et al., 1997]] Pimentel, D., Wilson, C., McCullum, C., Huang, R., Dwen, P., Flack, J. Tran, Q., Saltman, T., Cliff, T. (1997). Economic and environmental benefits of biodiversity. ''BioScience'', Vol. 47, No. 11., pp. 747-757.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/OverviewTeam:TU-Munich/Results/Overview2013-10-29T03:07:53Z<p>FlorianA: /* Results Overview */</p>
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== Results Overview ==<br />
Having spent our summer in the lab, we are proud to present our accomplishments. Upto the European regional jamboree in Lyon, we have created 72 BioBricks and devices, transformed and selected 20 different transgenic GM-mosses and characterized our effectors as recombinant proteins produced in ''E. coli'' and in our moss ''Physcomitrella patens'', a chassis newly introduced into iGEM. We took further steps to put our phytoremediation project into practice by developing concepts for the implementation of our PhyscoFilter in the environment and by analyzing the economic potential of this innovative technology in our Entrepreneurship section. Additional to our wetlab work, we contributed a very powerful software tool for the annotation of BioBricks from the Parts Registry and created tutorials to pass this summer´s experiences and skills on to the subsequent iGEM generations. <br />
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<div class="box-left overview"><html><img src="https://static.igem.org/mediawiki/2013/d/dc/TUM13_results-1.jpg" /></html><br />
===BioBricks===<br />
We created 72 BioBricks, including BioBricks advancing the use of ''Physcomitrella patens'' as a chassis, BioBricks enhancing phyto- remediation applications and BioBricks for the light triggered kill-switch mechanism.<br />
([https://2013.igem.org/Team:TU-Munich/Results/BioBricks Read more])<br />
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<div class="box-right overview"><html><img src="https://static.igem.org/mediawiki/2013/8/80/TUM13_results-2.jpg" /></html><br />
===Effector studies===<br />
We selected six different effector proteins and produced them in ''Escherichia coli'' and characterized them further concerning stability and activity.<br />
([https://2013.igem.org/Team:TU-Munich/Results/Recombinant Read more])<br />
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<div class="box-left overview"><html><img src="https://static.igem.org/mediawiki/2013/e/ee/TUM13_results-3.jpg" /></html><br />
===Moss Methods===<br />
We performed some general experiments to optimize ''Physcomitrella patens'' concerning tolerance of toxins, growth optimization and the use of different cultivation surfaces. Furthermore, we created 20 different strains of transformed moss during our visit to Prof. Reski´s lab in Freiburg. ([https://2013.igem.org/Team:TU-Munich/Results/Moss Read more])<br />
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<div class="box-right overview"><html><img src="https://static.igem.org/mediawiki/2013/e/e0/TUM13_Receptor_small.png" /></html><br />
===Localization===<br />
In order to ensure the best functionality for our effectors we designed and used several localization methods aswell as different verification techniques to see if our localizations were successful. <br />
([https://2013.igem.org/Team:TU-Munich/Results/Localization Read more])<br />
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<br />
<div class="box-left overview"><html><img src="https://static.igem.org/mediawiki/2013/2/2a/TUM13_results-4.jpg" /></html><br />
===PhyscoFilter===<br />
After selection and regeneration of the transgenic moss, we started the experiments with them and characterize their properties as PhyscoFilter. ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss Read more])<br />
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<br />
<div class="box-right overview"><html><img src="https://static.igem.org/mediawiki/2013/6/63/TUM13_project-6.jpg" /></html><br />
===Kill Switch===<br />
After selection and regeneration of the transgenic moss, we started the experiments with them and characterize their properties as PhyscoFilter. ([https://2013.igem.org/Team:TU-Munich/Results/KillSwitch Read more])<br />
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===Implementation===<br />
The creation of new environmental solutions doesn't stop at the development of GM moss. We tried to find out how a large-scale biofilter could be implemented.<br>[https://2013.igem.org/Team:TU-Munich/Results/Implementation Read more]<br />
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===AutoAnnotator===<br />
Protein coding BioBricks constitute large parts of the Parts Registry. We created a software for in-silico characterization of various para- meters summed up in a standardized table. To improve the parts registry, <nowiki>RFC 96</nowiki> proposes a range of characteristics determined by the AutoAnnotator.<br />
([https://2013.igem.org/Team:TU-Munich/Results/Software Read more])<br />
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===Entrepreneurship===<br />
To translate science into applied technology available to the public, economic and business factors play increasingly important roles. We took the first steps into this direction by examining criteria for implementation and possibilities of business models in biotechnology. ([https://2013.igem.org/Team:TU-Munich/Results/Economics Read more])<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/File:TEV_FRET_Reporter.pngFile:TEV FRET Reporter.png2013-10-29T03:05:19Z<p>FlorianA: uploaded a new version of &quot;File:TEV FRET Reporter.png&quot;</p>
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<div></div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Project/LocalisationTeam:TU-Munich/Project/Localisation2013-10-29T02:58:41Z<p>FlorianA: /* Verification of Localization &ndash; introducing a superior reporter protein */</p>
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== Localization in ''Physcomitrella patens'' ==<br />
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[[File:TUM13_Lokalisation_1.4.png|thumb|center|910px| '''Figure 1''': Overview of different possible localisations for effector proteins.]]<br />
In order to use ''P. patens'' as a chassis for Phytoremediaton, it is essential to be able to express effector proteins in different compartments. This includes '''cytoplasmatic expression''' of cytosolic effectors which degrade xenobiotics capable of crossing the cell membrane and which might depend on cofactors for degradation or conjugation. Secondly, there is the possibility to '''secrete effectors''' outside of the cell for an easy access to their respective target molecules. Other applications benefit from the expression of '''immobilized effectors''' on the inner or outer cellular membrane. This allows the creation of systems that do not release transgenic proteins into the environment and of systems that are able to internalize substances attached to recombinant binding proteins.<br><br />
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'''Cytosolic''' expression of effector proteins was achieved by cloning the respective BioBrick downstream of the Actin_5 promoter. For the '''secretion''' of effectors, we compared several signal peptides with a prediction software and chose the signal peptide from the SERK receptor of ''Physcomitrella patens''. Additionally, we tested the signal peptides of a secreted antibody from mouse [[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009 Gitzinger et al., 2009]] for comparison. For this purpose, the signal peptides were created as <nowiki>RFC 25</nowiki> N-parts for the creation of fusion proteins with effectors available in <nowiki>RFC 25</nowiki>. Finally, the '''immobilization''' of recombinant effector proteins on a receptor which is functional in ''Physcomitrella patens'' was investigated by the construction of a synthetic receptor based on the SERK receptor.<br />
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=== Cytoplasmatic Expression ===<br />
Cytoplasmatic expression of effector proteins is necessary for effectors that either depend on intracellular cofactors or for effectors with mechanisms linked to at least one exclusively intracellular component. For our project, this is the case for:<br />
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* Catechol-1,2-dioxygenase: Ferredoxins necessary to regenerate the oxygen-inactivated iron<br />
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* Glutathion-S-transferase: Glutathion necessary for coupling reactions<br />
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=== Secretory Expression ===<br />
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Effectors proteins containing '''disulfide bonds''' have to be secreted, as the reducing milieu of the cytosolic compartment does not allow formation of disulfid bonds. For secretion, the protein has to be fused to a '''signal peptide''' for the translocation into the endoplasmatic reticulum. Therefore we created two BioBricks encoding a signal sequences for endoplasmatic reticular translocation: The '''SERK signal peptide''' ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]) of the Somatic Embryogenesis Receptor Kinase from ''Physcomitrella patens'' (see [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions#Prediction_of_Signal_Peptides Prediction of Signal Peptides] for more details) and the '''IgKappa signal peptide''' of the Ig Kappa chain from ''Mus musculus'' ([http://parts.igem.org/Part:BBa_K1159304 BBa_K1159304]) [[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]]. Our following effector needs to be secreted:<br />
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* Laccase from ''Bacillus pumilus'': 1 disulfide bond<br />
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=== Membrane-bound Expression ===<br />
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[[File:TUM13_SERK-Receptor.png|thumb|right|300px| '''Figure 2''': Schematic design of an effector immobilized on a transmembrane domain.]]<br />
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For '''substance binding proteins''' or as an '''alternative to secretion''' for degrading enzymes, it is convenient to anchor effectors on the plant´s surface inside its cytoplasmatic membrane.<br />
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For this purpose, we added a '''signal for secretion''' at the N-terminus of our effector protein as well as a downstream '''transmembrane domain''' from ''Physcomitrella patens'' ([http://parts.igem.org/Part:BBa_K1159315 BBa_K1159315]) for membrane localization. Furthermore, we fused GFP ([http://parts.igem.org/Part:BBa_K1159311 BBa_K1159311]) to the intracellular site of the transmembrane region for the verification of membrane localization via fluorescence microscopy. We set a '''linker''' containing ''Strep''-tag II and a TEV cleavage site in between the effector protein and the transmembrane region. This allows us to characterize the membrane-bound effectors by incubating the plant with TEV Protease and thus releasing the respective effectors. Hence, purification via the ''Strep''-tag II and successive characterization of the effector is possible.<br />
<br />
For more details on the design process of the transmembrane region, see [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions Protein Predictions].<br />
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==Modularization: Post-translational fusion using SpyTag&nbsp;&&nbsp;SpyCatcher==<br />
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[[File:SpyCatcherSpyTag.png|thumb|center|910px|'''Figure 3''': Steps of the localization process via SpyTag and SpyCatcher.]]<br />
To create transgenic moss that is able to degrade customized combinations of xenobiotics targeted by different effector molecules, we introduced the SpyCatcher/SpyTag system into our project [[http://www.pnas.org/content/109/12/E690 Bijan Zakeri et al., 2012]]. This system was created for '''posttranslational protein fusion''' based on a covalent bond which is formed between the side chains of residues of SypCatcher and SpyTag. SpyCatcher and SpyTag were created by splitting and engineering the FbaB domain from ''Streptococcus pyogenes''. The splitted parts recognize each other and form a '''covalent isopeptide bridge''', like in their natural non-splitted form.<br />
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Recombinant effector proteins fused to the SpyTag can thus be '''expressed separately''' (e.g. at a different intensity, via a secretory pathway) from receptors fused to the SypCatcher. This enables us to control the SERK-receptor´s expression through a strong promoter while individually adjusting the expression of different effector proteins according to their respectively desired concentration (see Fig. 3.2).<br />
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Another application lies in the '''fusion of multimeric effector proteins to receptors''', where the fusion of all individual subunits to the receptor is not a viable option due to '''steric reasons'''. The SpyCatcher/SpyTag system bypasses this problem, as it allows the multimeric protein to assemble into its active form before its immobilization on the exterior membrane by a respective receptor (see Fig. 3.3).<br />
<br />
But the biggest advantage of the system is that it allows you '''non-canonical post-translational protein fusions'''. With this system '''you can fuse e.g. the C-terminus of a protein not only with the N-terminus of another protein but also the C-terminus.''' The reason is that the formation of the covalent bond is not a canonical peptide bond but a isopeptide bond between the side residues of SpyCatcher and SpyTag.<br />
<br />
It also allows you to create membrane-bound enzyme assembly line for two-step catalyzed reactions by fusing the two involved enzymes to the both termini of SpyTag.<br />
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[[File:SpyCatcherSpyTag_assembly_line.png|thumb|center|910px|'''Figure 4''': Steps of the localization process of a fusion protein consisting two enzymes via SpyCatcher/SpyTag system, resulting in a branch structure.]]<br />
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==Verification of Localization &ndash; introducing a superior reporter protein==<br />
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[[File:TUM13_Annimated_test.gif|thumb|right|300px| '''Figure 5:''' Homologous structure (3ppt_A) of the NanoLuc Luciferase]]<br />
To analyse the proper localization of our moss constructs we created a [http://parts.igem.org/wiki/index.php/Part:BBa_K1159001 BioBrick BBa_K1159001] from the '''NanoLuc''' Luciferase by Promega. NanoLuc Luciferase can be up to 240x brighter than conventional firefly luciferase and is at least two times smaller than other luciferases. This makes the NanoLuc Luciferase an '''ideal reporter protein''' which can not only be used to verify proper localisation, but can also be employed whenever a low detection threshold is needed.<br />
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==References:==<br />
[[http://www.plantphysiol.org/content/127/4/1430 Schaefer and Zryd, 2001]] Schaefer, D.G. and Zrÿd, J. (2001). The Moss ''Physcomitrella patens'', Now and Then. ''Plant Physiology'', 127(4):1430-1438.<br><br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315829/ Hagan et al., 2010]] Hagan RM, Björnsson R, McMahon SA, Schomburg B, Braithwaite V, Bühl M, Naismith JH, Schwarz-Linek U. (2010). NMR spectroscopic and theoretical analysis of a spontaneously formed Lys-Asp isopeptide bond. ''Angew Chem Int Ed Engl''. 2;49(45)<br><br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Gitzinger M, Parsons J, Reski R, Fussenegger M (2009). Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. ''Plant Biotechnol J''. 7(1):73-86.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:46:24Z<p>FlorianA: /* Implementation */</p>
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==Our Project for this summer: Remediation.==<br />
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<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and to bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
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==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
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==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed and the formation of isopeptide bonds were confirmed by pull-down experiments and reducing SDS-PAGE. Summarizing these results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a new chassis.<br />
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==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick '''Erythromycin Esterase (EreB)''' which degrades macrolide antibiotics. Additionally we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular part of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life in river water was estimated. All data was fitted in our enzyme kinetic modeling. The aim was to analyze this data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football fields would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
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==BioAccumulation==<br />
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Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
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==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy and safety. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisory board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a large area of contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as the laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries with highly contaminated waters. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein into the water to degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure&nbsp;3).<br />
<br />
==Supporting the iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years. We can proudly say that we invested efforts to take the iGEM community to another level. We created a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and compiles alignments with data from various data banks. In the end all collected information for a BioBrick is exported into a standardized table which can easily be integrated into the part descriptions of BioBricks. We submitted a RFC for the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96]. Besides this software toll we have written tutorials on wiki programming, creating animated gifs of protein structures and the usage of Arduino microcontrollers for future iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:46:06Z<p>FlorianA: /* Implementation */</p>
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==Our Project for this summer: Remediation.==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white; float: right;" src="http://player.vimeo.com/video/76195786" width="400" height="240" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and to bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed and the formation of isopeptide bonds were confirmed by pull-down experiments and reducing SDS-PAGE. Summarizing these results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a new chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick '''Erythromycin Esterase (EreB)''' which degrades macrolide antibiotics. Additionally we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular part of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life in river water was estimated. All data was fitted in our enzyme kinetic modeling. The aim was to analyze this data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football fields would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white;float: right;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
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Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy and safety. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisory board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a large area of contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as the laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries with highly contaminated waters. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein into the water to degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==Supporting the iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years. We can proudly say that we invested efforts to take the iGEM community to another level. We created a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and compiles alignments with data from various data banks. In the end all collected information for a BioBrick is exported into a standardized table which can easily be integrated into the part descriptions of BioBricks. We submitted a RFC for the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96]. Besides this software toll we have written tutorials on wiki programming, creating animated gifs of protein structures and the usage of Arduino microcontrollers for future iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:45:30Z<p>FlorianA: /* BioDegradation */</p>
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==Our Project for this summer: Remediation.==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white; float: right;" src="http://player.vimeo.com/video/76195786" width="400" height="240" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and to bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed and the formation of isopeptide bonds were confirmed by pull-down experiments and reducing SDS-PAGE. Summarizing these results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a new chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick '''Erythromycin Esterase (EreB)''' which degrades macrolide antibiotics. Additionally we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular part of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life in river water was estimated. All data was fitted in our enzyme kinetic modeling. The aim was to analyze this data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football fields would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white;float: right;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy and safety. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a large area of contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as the laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries with highly contaminated waters. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein into the water to degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==Supporting the iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years. We can proudly say that we invested efforts to take the iGEM community to another level. We created a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and compiles alignments with data from various data banks. In the end all collected information for a BioBrick is exported into a standardized table which can easily be integrated into the part descriptions of BioBricks. We submitted a RFC for the [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Besides this software toll we have written tutorials on wiki programming, creating animated gifs of protein structures and the usage of Arduino microcontrollers for future iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:44:01Z<p>FlorianA: /* Our Project for this summer: Remediation. */</p>
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==Our Project for this summer: Remediation.==<br />
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<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and to bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed and the formation of isopeptide bonds were confirmed by pull-down experiments and reducing SDS-PAGE. Summarizing these results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a new chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick '''Erythromycin Esterase (EreB)''' which degrades macrolide antibiotics. Additionally we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular part of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life in river water was estimated. All data was fitted in our enzyme kinetic modeling. The aim was to analyze this data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football field would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white;float: right;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy and safety. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a large area of contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as the laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries with highly contaminated waters. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein into the water to degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==The iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years and we also invested some effort to advance the iGEM community further. We programed a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and does alignments with various data banks. In the end all collected information for a BioBrick is collected in a standardized table which can easily be integrated into the part description of a BioBricks. We submitted a RFC for this [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Beside this software toll we have written tutorials on wiki programming, creation of animated gifs of protein structures and the usage of Arduino microcontrollers for iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/HumanPractice/EducationTeam:TU-Munich/HumanPractice/Education2013-10-29T02:39:40Z<p>FlorianA: /* Introducing Synthetic Biology Kits at educational institutions */</p>
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== Introducing Synthetic Biology Kits at Educational Institutions ==<br />
<br />
What is better than teaching one class about SynBio?<br><br />
Enabling many teachers to complete this task!<br />
<br />
<div class="quote"><br />
"If you want to build a ship, do not drum up people together to collect wood and do not assign them tasks and work, but rather teach them to long for the endless immensity of the sea."<br />
<div class="author">- Antoine de Saint-Exupéry</div><br />
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==Introduction==<br />
[[File:TUM13_Schulkits.jpg|thumb|right|400px|'''Figure 1:''' Prepared Educational-Kits, ready for delivery!]]<br />
Following this famous statement in pedagogics, we realized the importance to inspire and teach students in educational institutions to long for the endless immenseness of Synthetic Biology. We want to make it possible for disciples to take their first steps in the enormously evolving field of Synthetic Biology and let them take a breath of this subject, which will change the world in the near future due to its unlimited fields of application.<br />
<br />
The idea is to send an "Educational-Kit" with all required reagents and lab-protocols to educational institutions. Based on the experiments we designed we'd like to inspire young prospective people to think about a promising career in science. Our experiments are styled constitutive, based on every previously performed experiment, so every student, independent of training level, is able to understand and take notice of the resulting phenomena enabled by Synthetic Biology.<br />
<br />
==The Educational-Kit==<br />
The reagents, which are necessary for the realization of the experiments are all found in the kit, except for competent cells, which have to be stored at -20°C. It was set much value on the autonomous preparation of the final reagents, so nearly all reagents will be sent in their blank shape. Our aim is teaching the students all, or at least most, of the necessary steps of the designed experiments during the execution. The kit consists of the following reagents:<br />
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{|cellspacing="0" border="1" right <br />
|+ '''Table 1:''' Contents of the educational kit<br />
! align="center";| Reagent<br />
! align="center";| Amount of the Reagent<br />
! align="center";| Is it existent in the kit?<br />
|-<br />
|LB-Medium<br />
| align=right | Reagents for about 3 L are in the kit<br />
| align=center | Yes<br />
|-<br />
|"Banana-Odor"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|"RFP-Generator"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|"Luciferase"-Plasmid<br />
| align=right | 100 µl (10 ng/µl)<br />
| align=center | Yes<br />
|-<br />
|Isoamylalcohol (>98 %)<br />
| align=right | 10 ml<br />
| align=center | Yes<br />
|-<br />
|Agar plates (Resistance: Kanamycin)<br />
| align=right | 4 Plates<br />
| align=center | Yes<br />
|-<br />
|Agar plates (Resistance: Chloramphenicol)<br />
| align=right | 2<br />
| align=center | Yes<br />
|-<br />
|L-Arabinose (500 mM)<br />
| align=right | 3.76 g (50 ml)<br />
| align=center | Yes<br />
|-<br />
|Kanamycin<br />
| align=right | 25 ml<br />
| align=center | Yes<br />
|-<br />
|Chloramphenicol<br />
| align=right | 25 ml<br />
| align=center | Yes<br />
|-<br />
|Competent Cells <br />
| align=right | 6 Tubes (Aliquotvolume: 150 µl)<br />
| align=center | No<br />
|-<br />
|} <br />
<br />
As you may have noticed, we provide three different plasmids, which are used separately in each designed experiment. After the experiments have been performed, disciples should have experienced the whole way from transformation, over cultivating cells, inducing the production of proteins. In the end they should also have observed the conversion of a substrate by the produced enzyme.<br />
<br />
==Experiment Number 1: Bacteria in Red==<br />
[[File:TUM13_RFP_Generator.jpg|thumb|right|400px|'''Figure 2:''' Results of Experiment Number 1]]<br />
The aim of the first experiment is to transform the [http://parts.igem.org/Part:BBa_K801100 "RFP-Generator"-Plasmid] into ''Escherichia coli'', which contains genes, coding for Chloramphenicol resistance, plus a red fluorophor. Then they should be plated on Chloramphenicol-Agarplates. The Agarplates, provided with the antibiotica Chloramphenicol, trigger a selection process, whose aim is to kill all bacteria which have not incorporated the "RFP-Generator"-Plasmid. The expression rate of the red fluorophor is controlled by a constitutive promoter, so the '''R'''ed '''F'''luorescent '''P'''rotein is expressed continuously. After just about one day of incubation time, the students can notice a change in color of the bacteria from ocher to red with the naked eye.<br />
<br />
The Biobrick we used to realize this experiment was firstly designed by the [https://2010.igem.org/Team:Groningen iGEM Team of Groningen in 2010] to simplify the cloning step by detecting internalized plasmids, which self-circularized without integration of the desired insert. Bacteria, which internalize self-circularized Plasmids will produce red colonies, so these undesired bacterial colonies can be avoided in the next steps of the experiment, to be sure of using the right construct. <br />
<br><br />
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<br />
==Experiment Number 2: Glowing Bacteria Lamp==<br />
[[File:TUM13_Luciferase.jpg|thumb|right|300px|'''Figure 3:''' Structure of the Luciferase]]<br />
In the second experiment students should perform the transformation, which they learned in experiment one, with the "Luciferase"-Plasmid. The Plasmid encodes genes for Kanamycin resistance and a lux - gene cassette containing a protein-fluorophor called Luciferase. Another very important point is that the lux-gene is subordinated to a L-Arabinose Operon. After the transformation of the DNA-Plasmid has taken place, students should learn how to cultivate bacteria in liquid LB-Medium and how to induce protein expression by using L-Arabinose as an inductor of the L-Arabinose promoter. The glow of the Luciferase can already be observed after a few hours.<br />
<br />
==Experiment Number 3: Banana Odor Generator==<br />
[[File:SchmolkerMollath.png|thumb|right|400px|'''Figure 4:''' Results of Experiment three]]<br />
In the third experiment, all aspects learned in the previous experiments come together. The [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] encodes an enzyme called Alcohol-Acetyltransferase I. This enzyme is able to convert Isoamylalcohol into Isoamylacetat, which spreads the odor of banana. Additionally the plasmid harbors a resistance gene for Kanamycin and an inducible L-Arabinose promoter, which controls the expressions rate of the Alcohol-Acetyltransferase I enzyme. The students should transform the [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] into ''Escherichia coli'', cultivate the successfully transformed bacteria in a larger scale and in the end, induce the protein expression by using L-Arabinose. In the last step, the students should add the substrate Isoamylalcohol to the cell culture, so the produced enzyme can transform it into Isoamylacetat. <br />
<br />
The Biobrick, which spreads the odor of banana, after transforming its substrate, was first established by the [http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team of the Massachusetts Institute of Technology in 2006]. Their aim was to produce different compounds in ''Escherichia coli'' that smell fragrant. Another very interesting application of the "Banana-Odor" Biobrick is pursued by the [https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team of Queens in 2013]. This team tries to neutralize foot odor by creating a skin creme containing bacteria with a genetically engineered metabolic pathway to neutralize the volatile compounds that cause these smells. This pathway begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present in foot odor, and converts it into banana smell. By establishing this skin creme, it should be possible to prevent people from being attacked by mosquitos and saved from typical alienable diseases mosquitos spread.<br />
<br />
==Target Audience==<br />
The target audience for our kit are secondary schools with focus on a biotechnological educational pathway, so all students have access to the required laboratory equipment for the experiments. Before sending the kits to the schools, we tested them in detail - with success. All schools we contacted to spread our Synthetic Biology kits are very enthusiastic concerning our idea of supporting young prospective scientists, so they are very excited to receive and use our kit!<br />
<br />
==Availability of the School-kit==<br />
We appreciate other interested schools in trying out our “School kits”. Do not hesitate contacting us. We are happy to supply you with our forward-looking Synthetic Biology “Educational-kit”!<br />
<br />
You can '''send requests''' to: igem@wzw.tum.de<br />
<br />
Due to the World Championship Jamboree of iGEM, we offer the description of our "Educational Kit" directly to everyone, who is interested in it! Just click on the download-Button to get it! This decision supports our open-source idea, to facilitate the education of every pupil, prospective scientist and scientists. <br />
<br />
[[media:Educational_kit.pdf|'''Get your Educational kit!''']]<br />
<br />
We are very sad to tell you that our "Educational Kit" is just available in german because our target audience have been german secondary schools with a focus on biotechnological educational pathway. <br />
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==References==<br />
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[[https://2010.igem.org/Team:Groningen iGEM Team Groningen 2010]] <br><br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/22368493 Close D et al., 2012]] Close D, Xu T, Smartt A, Rogers A, Crossley R, Price S, Ripp S, Sayler G (2012). The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter. ''Sensors'', 12(1):732-52. <br><br />
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[[http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team Massachusetts Institute of Technology 2006]] <br><br />
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[[https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team Queens 2013]] <br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/HumanPractice/InterviewsTeam:TU-Munich/HumanPractice/Interviews2013-10-29T02:36:47Z<p>FlorianA: </p>
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==Expert Counsel: An overview==<br />
All throughout the development process of our PhyscoFilter we got in touch with experienced experts and stakeholders to constantly improve our technical solution and to assess potential users demands on its properties.<br />
<br />
[[File:TUM13_Spread_the_word.png|thumb|center|910px|'''Figure 1:''' Overview of our interviews]]<br />
<br />
==1) 4<sup>th</sup> Water Research Horizon Conference “Water Scarcity and Global change” in Berlin==<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference.JPG|thumb|right|350px|'''Figure 2''': 4th Water Research Horizon Conference “Water Scarcity and Global change” in Berlin]]<br />
The main goal of the participation on the 4th [http://www.ufz.de/water-research-horizon Water Research Horizon Conference] “Water Scarcity and Global change” in Berlin on the 25th of June 2013 was to inform ourselves about worldwide water scarcity and global change. We hoped to get inspirations for our '''outreach''' and '''human practice''' effort. Moreover we were interested in the worldwide practical realization of minimizing “the water problem”. Especially the diverse ways of approach in different countries and the existing solutions were of interest (see Figure 2) to see how our project fits into this scope. In addition to the main speeches of ambassadors from different communities and companies we participated on a workshop session with speeches and discussion called ''“Water and Soils for Food and Energy - A Question of '''Technological Development'''?”''. <br />
<br />
We realized that it is quite difficult to give a straight answer to this question. J. Bouma (formerly Wageningen University, The Netherlands) answered with ''“No, not alone, but start with existing technology and go from there”''. Whereas R. Ardakanian (United Nations University, Dresden) just said ''“yes”'' after his speech. But not only the answers on the topic question differed, also the point of view concerning to the worldwide water problem, scarcity and the definition of common water diverged. In his speech, R. Ardakanian placed '''water''' in context with '''waste'''. He said that waste can be a resource for the production of food and energy and must contribute as '''recycled''' waste in technology. According to E. Adar the goal and challenge for the next generation is (Ben Gurion University, Negev, Israel) minimizing the water use and costs. To him it was evident that water is a synonym for food and a commodity, not a resource like gold.<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference_speech_1.JPG|thumb|left|350px|'''Figure 3''': speech about ''"historic and future variations in global water"'' from J. Sheffield]]<br />
<br />
Furthermore M. Lange (The Cyprus Institute, Nicosia) presented his speech titled ''"Ways about Climate Change and Energy-Water Nexus in the Mediterranean"''. A. Porporato (Duke University, NC, USA) gave a lecture on the role of '''ecohydrological variability''' in productivity and sustainability of ecosystems.<br />
<br />
The following general speeches about the water scarcity and climate change, informed the audience about different present projects to reduce the water scarcity. G. Teutsch (UFZ Leipzig) who opened the conference talked about a ''“quick start”'' to reduce the water scarcity, which sticks together with the urgent topic of '''climate change'''. The main statement of the second speaker's speech ,P. Krebs (TU Dresden), was that not visions yield viable results, but only excellent '''concepts''', new '''projects''' and '''community action'''. That the water topic has also an important role in politics, was approached from F. Holzwarth, (BMU Bonn) whose goal is forming a ''“water alliance with new individual faces”''. He stated that in contrast to the climate change, the water scarcity isn’t a major topic in politics, because ''“disasters drive '''politics'''”''. In his speech U. Weber (DFG Bonn) picked out science (project, implementation and ideas) and society (public, politic) in relation to water scarcity and climate change as central themes. In turn, J. Sheffield (Princeton University, USA; see Figure 3) broached the issues of the transition between drought and floods.<br />
<br />
[[File:TUM13_Water_Research_Horizon_Conference_speech_2.JPG|thumb|right|350px|'''Figure 4''': speech about addressing our emerging water futures:The water-agriculture-energy-ecosystem nexus from G.Bidoglio]]<br />
The theoretical aspect of controlling natural water's quality in a '''mathematical model''' was presented by G. Bidoglio(EC-JRC IES Ispra, Italy; see figure 4). Implemented Projects are the Joint Research Project SMART (integrated Water Resource Management in the Lower Jordan Valley) and the Guanting Project (sustainable water and agricultural land use in the Guanting watershed under limited water resources).<br />
<br />
In summary the visit of the conference made us realize that water '''scarcity''' and '''water quality''' are in fact vital problems that already attract much attention in academic circles. In contrast, we did not know that the global water situation is such a multifaceted subject and we were stunned by the broad range of subjects that were covered during the conference. As the conference took place at the beginning of our project, the impressions we received from the conference helped us '''understanding''' what problems we should address when talking to a broader audience in our subsequent human practice efforts.<br />
Moreover the presentation of currently happening innovative projects '''inspired''' us in the design of our filter prototypes.<br />
<br />
==2) Workshop "Drugs in the Environment" in Berlin organized by the RCDS==<br />
[[File:TUM13_RCDS-conference.png|thumb|right|200px|'''Figure 5''': Description]]<br />
At the end of May we joined the "Drugs in the environment" conference that was organized by the [http://www.rcds.de/ RCDS] (which is the youth organisation of the CDU, the currently governing political party in Germany) and the [http://www.vfa.de/ German Association of Research-Based Pharmaceutical Companies].<br> <br />
During this discussion meeting we had the opportunity to meet and discuss with several experts as well as with representatives of leading German pharmacy companies such as Bayer and Novartis. The main focus of this discussion was on the point whether '''pollution''' of the environment by '''pharmaceutic substances''' is a considerable problem for our society. Furthermore whether Germany is going to '''invest money''' to reduce this pollution and who is responsible for financing these measures. The conclusion of this conference was that in Germany there is no acute danger for mankind by contamination of the environment by pharmaceutic substances. In contrast the lowering of the detection limit for several substances leads to an '''awareness''' that there is contamination caused by mankind and that several people are not willing to accept this pollution.<br />
<br />
==3) Biotechnology 2020+ Conference of the Ministry of Research and Education in Berlin==<br />
<br />
[[File:TUM13_Biotechnology 2020+.jpg|thumb|right|350px|'''Figure 6''': Biotechnology 2020+ Conference of the Ministry of Research and Education: first discussion round about the topic “result of the previous activities and recommendations for action for the continuation of the initiative "Biotechnology 2020+ conference" with the five ambassadors of '''five research institutions''' ( Frauenhofer Society, Helmholtz Association, Max-Planck Society, Leibniz Association, TU Braunschweig)]]<br />
For the third year in a row the German Ministry of Research and Education invited all German iGEM Teams to join the "[http://www.biotechnologie2020plus.de Biotechnology 2020+ conference]" in Berlin on June 27th. ''“Which products and biotechnological processes are fundamental? “'', ''“How will the next generation accept the biotechnological processes?”'' and ''“Which projects are already initiated?”''. Experts in the fields economy, politics and science and the German iGEM teams came together at the conference and tried to answer these questions. In his speech, Dr. Helge Braun (parliamentary State Secretary of the BMBF) emphasized that a creative teamwork with an own developed ''"language"'' is necessary. He introduced the new biotechnological ideas: biological implants, biomimetic desalination plants and microbial fuel cells. In the first discussion round about the topic “result of the '''previous activities''' and '''action recommendations''' for the continuation of the initiative "Biotechnology 2020+ conference", five ambassadors of five research institutions (Frauenhofer Society, Helmholtz Association, Max-Planck Society, Leibniz Association, TU Braunschweig) (see figure 5) presented their '''recently''' implemented '''ideas''': printable biotechnology, cell-free bioproduction and synthesis of novel therapeutics. Exemplarily Prof. Dr. H. Jäckle (Max-Planck-Community) introduced the research network for Synthetic Biology MaxSynbio. The second discussion treated the subject "Open Source in the Biotechnology”. After the presentation there were workshops on the topic “On the way to a Biotechnology 2020+”. Additionally the German iGEM projects 2013 were exhibitied (see Figure 6) and the five research institutions presented their recent research projects.<br />
[[File:TUM13_Biotechnology 2020+ iGEM German teams.jpg|thumb|left|350px|'''Figure 7''': Biotechnology 2020+ Conference of the Ministry of Research and Education: exhibition of the German IGEM projects 2013]]<br />
<br />
In summary the participation informed us about the practicable operated '''novel biotechnology ideas'''. It enabled our cooperations and gave us an '''insight''' into projects from market-leading research institutions in Germany. Furthermore we got in contact with other iGEM teams which paved the way for an iteration of the German iGEM day. <br />
<br />
[[File:TUM13_Biotechnology 2020_Poster.png|thumb|right|300px| '''Figure 8''': Poster presentation of our PhyscoFilter. ]]<br />
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==4) Visit of the Helmholtz Center for Environmental Research (UFZ) in Leipzig ==<br />
<br />
[[File:TUM13_Visit of the UFZ.JPG|thumb|right|350px|'''Figure 9''': Visit of the UFZ: Johanna and Andreas from iGEM TU Munich team with Dr. Kuschk (left) and Dr. Müller (right)]]<br />
When we visited the Helmholtz Zentrum of Leipzig [http://www.ufz.de/index.php?en=2540 UFZ] on the 26 th of June 2013, we had three objectives. We wanted to '''present our project''', get another impartial '''opinion''' concerning our project idea and project implementation and lastly acquire '''general information''' about practical phytoremediation. Dr. Kuschk and Dr. Müller are chair members of the environmental biotechnology section and attend to '''phytoremediation''' or rather bioremediation (see figure 6). Their research focus lies in the area of zoonosis that means the interplay between plants and microorganisms. The plant acts with the root system as a carrier to quantify the microorganisms which dispose nitrate, sulfur or toluol. According to Dr. Kuschk genetic engineering work isn’t possible at the UFZ, because of the few personnel at the moment.<br />
<br />
<br />
Dr. Müller gave us a guided tour through the laboratories. (see Figure 7) Later on we had two conversations with experts on different issues concerning our project. Dr. Müller gave us very helpful annotations about our '''effectors'''. We were explained the sense of '''amplifying the resistance gene EreB''' in environment in relation to the biotechnological benefit. He informed us about the costs of blue light. Furthermore he alluded that we had to consider the '''enzyme specification''' in order to find the benefit of extincting erythromycin to clean water. Another point was the use of '''cathecholdioxygenase'''. According to Dr. Müller, depletion of catechol through microorganisms is a very effective process. So our next step was to show the efficiency of the depleting catechol through moss. <br />
<br />
[[File:TUM13_Visit of the UFZ laboratories.JPG|thumb|left|350px|'''Figure 10''': Visit of the UFZ: Dr. Müller leads two iGEM TU Munich team members through the laboratories.]]<br />
<br />
The main topic we discussed with Dr. Kuschk was the application of our project idea in '''practice''', outside the competition framework. Dr. Kuschk was in '''doubt''' about the practical implementation. His assessment was based on his comprehensive experiences with concepts and conversions he developed. In his opinion the '''costs''', the low concentration of polluting substances and the small contact surface have negative effects on the projects application. When it comes to the immobilization of substances, he prefers biofilms to membrane bounds. He also criticized our choice of degraded substances. In his opinion fecal substances are the cause of the high death and diseases rate in less developed countries. We explained that we chose harmful substances, according to the WHO, to establish our filter, as we'd like to show the '''degradation and bioaccumulation''' of them. In general he considered our project as basic research and not as practical application.<br />
In summary the visit of the UFZ enriched our knowledge in the issue of molecular genetics and gave us an idea how further project thoughts could look like. The guided tour through the laboratories of UFZ showed us '''execution examples''' of working phytoremediation concepts.<br />
<br />
==5) Expert Box: [http://www.pieris-ag.com Pieris AG]==<br />
[[File:TUM13_Expert_pieris.png|thumb|left|350px| '''Figure 11''': Expert interview with the [http://www.pieris-ag.com Pieris AG]]]<br />
The [http://www.pieris-ag.com Pieris AG] is a '''biopharmaceutical company''' founded in 2001 which is commercializing the '''anticalin''' production technology. Anticalins are designed to '''bind proteins''' that are a '''promising alternatives to antibodies'''. These are mainly used in red biotechnology. They are, for example, used in tumor therapy. As these engineered binding proteins can be directed against nearly every chemical structure, they are also an interesting tool for phytoremediation. Our idea was to apply the successful anticalin technology in a totally different context, namely for the '''detoxification of aquatic ecosystems'''. We contacted the Principal Scientist of the [http://www.pieris-ag.com Pieris AG] and they are confident that a meeting will be possible until the regional Jamboree in Lyon.<br />
<br />
==6) Expert Box: Prof. Reski (Freiburg University)==<br />
[[File:TUM13_expert_Reski.png|thumb|450px|left| '''Figure 12:''' Expert interview with Prof. Reski. ]]<br />
When we had chosen phytoremediation as our topic for this year's competition it became clear soon that ''Physcomitrella patens'' is a '''great chassis''' which could bring iGEM closer to the real world applications we are looking for. For this reason we contacted Prof. Dr. Reski who is a worldwide expert on ''Physcomitrella patens'' and especially its biotechnological application. He was very kind and liked our idea from the first moment on. Especially when we told him to introduce '''his ''Physco'' '''to iGEM. So he offered us help where ever we would need it. In the end we traveled five times to Freiburg (350 km) with different team members to perform the transformations in his lab. '''Fetching plasmids''' which are known to be functional in ''Physcomitrealla patens'' or just to discuss with him or his co-workers. We could win him as an '''advisor''' for our team. That is especially pleasant as iGEM starts bringing together universities and scientific groups from different places and and our iGEM team was not restricted to our own university.<br />
<br />
==7) Visiting the iGEM Team Freiburg 2013==<br />
[[File:Muenchen-Freiburg-2013.jpg|thumb|left|350px|'''Figure 5:''' iGEM Teams Freiburg and Munich joint during our first visit]]<br />
[[File:TUM13_Freiburg.JPG|thumb|right|200px|'''Figure 6:''' Our second visit]]<br />
During our transformation experiments at Prof. Reski's lab we spent more than one evening with the iGEM team Freiburg and had for example a very delicious barbecue and an interesting scientific exchange. Thank you a lot for your hospitality, congratulations for your success in Lyon and see you in '''Boston'''!<br />
<br />
==8) Expert Box: Greenovation (Freiburg)==<br />
<br />
[[File:TUM13_expert_Greenovation.png|thumb|450px|left| '''Figure 13:''' Expert interview with Dr. Schaaf, Principal Scientist at [http://www.greenovation.com/index.html Greenovation BioTech GmbH]]]<br />
In September we went on a road trip to visit interesting companies and experts to get some consultation on our plans. As there is a biotech company which successfully uses ''Physcomitrella patens'' as a '''production organism''' for '''therapeutic proteins''' it became clear that we had to meet a responsible person of this company. Dr. Schaaf, the Principal Scientist of [http://www.greenovation.com/index.html Greenovation] invited us for a discussion and subsequently he showed us the production facility and the other labs of Greenovation. During the meeting we discussed their work flow for ''Physcomitrella'' and ours. We talked about '''production rates''' of therapeutic proteins and learned about its '''cultivation conditions''' in a bioreactor as well as in nature. During the discussion we also considered ordering the transformation of PhyscoFilter plants (so calles BryoTechnology). This '''hypothetical collaboration''' would have been possible and would have cost us around 50,000 to 70,000€ per effector. The price would have included the '''DNA preparation''', '''transformation''' and '''expression analysis''' of 1000 clones. Beside the possibility of a collaboration with PhyscoFilter the company also holds '''patents''' which could, in theory, block the application of ''Physcomitrella'' for commercial phytoremediation. In this case it would become necessary to buy a license for the usage of transgenic ''phycomitrella'' plants. Other experts we have consulted can be found in our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews Advisory Board].<br />
<br />
==9) Expert Box: Prof. Posten (KIT Karlsruhe)==<br />
During our road trip we talked to Prof. Dr.-Ing. Clemens Posten, who is head of the [http://bvt.blt.kit.edu/ Institute of bioprocess engineering] at the Karlsruhe Institute of Technology (KIT). During our visit we were shown the institutes's bioreactors and Prof. Posten gave us an idea of how a '''symbiosis between plant and technology''' can look like. In the past his group worked on a collaboration project with Prof. Dr. Reski (see our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews Advisery Board]) on biological process engineering for ''Physcomitrella patens''. As part of this discussion we figured out several important parameters to control and possible problems to solve in order to successfully implement our ''PhyscoFilter''. <br><br />
Read more about the results of this [https://2013.igem.org/Team:TU-Munich/Project/Implementation consultation].<br />
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==10) Visiting the iGEM Team Heidelberg 2013==<br />
On our road trip we also spent an evening with the iGEM team '''[https://2013.igem.org/Team:Heidelberg Heidelberg 2013]'''. At first they told us that they were too busy with experiments but in the end they found 3 team members who showed us their lab and the gorgeous city of Heidelberg. Thank you a lot and see you in '''Boston'''!<br />
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==11) Expert box: Bestmann Green Systems (near Hamburg)==<br />
[[File:TUM13_expert_Bestemann.png|thumb|350px|left| '''Figure 14:''' ]]<br />
[http://www.bestmann-green-systems.de/ Bestmann Green Systems] is an engineering company which offers commercial solutions for '''embedding vegetation in aquatic ecosystems'''. As this is a major point of our implementation project we also talked to this company (because of the actual distance a visit was not possible). This company also sells '''swimming raft''' devices which allow the cultivation of plants on free water. This technology was highly interesting also for our [https://2013.igem.org/Team:TU-Munich/Results/Economics business model] in which [http://www.bestmann-green-systems.de/ Bestmann Green Systems] could be a potential collaboration partner.<br />
<br />
==12) Discussion of our Project with Dr. Christoph Then from Testbiotech==<br />
Dr. Christoph Then is a recognized expert on '''risk assesment''' in the biotechnological field and the CEO of the [http://www.testbiotech.org/ Testbiotech registered association]. Amongst many other engagements, he has worked as an expert advisor on agriculture and genetic engineering for the '''Green Party''' in the Bavarian Landtag (legislative assembly in a German state) and as expert for '''Greenpeace on agriculture, genetic engineering and consumer affairs'''. Since our iGEM project deals with Green Biotechnology and since our '''various implementation ideas include biosafety and GMO release aspects''', he was the ideal expert to talk to. We had a very productive and interesting discussion that helped us get a much clearer picture on these aspects as well as on the '''regulations of GMO authorisation'''.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-MunichTeam:TU-Munich2013-10-29T02:26:59Z<p>FlorianA: </p>
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== PhyscoFilter &ndash; Clean different. ==<br />
The contamination of aquatic ecosystems with a multitude of anthropogenic pollutants has been a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation ([https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation]) or specific binding ([https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]) of pollutants. The autotrophic, sedentary, aquatic nature of the moss [https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] makes it an optimal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] prevents unintended environmental spreading by limiting viability to places where the spectrum of sunlight is appropriately filtered. Furthermore, we have developed a device to [https://2013.igem.org/Team:TU-Munich/Results/Implementation implement our filter] in an aquatic environment, investigated the application of this new technology and examined its [https://2013.igem.org/Team:TU-Munich/Results/Economics economic feasibility]. Based on our results, the PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.<br />
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== Achievements ==<br />
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At the European Jamboree our team achieved the following:<br />
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*We won a gold medal.<br />
*We advanced to the World Championship Jamboree.<br />
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*We were the first runner up in the undergrad section.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Project/PhyscomitrellaTeam:TU-Munich/Project/Physcomitrella2013-10-29T02:26:09Z<p>FlorianA: /* General description */</p>
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==''Physcomitrella'' - A new chassis for iGEM==<br />
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===General description===<br />
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The moss ''Physcomitrella patens'' belongs to the land plant division Bryophyta, which are one of the earliest representatives of the land plants (Embryophyta) having evolved from green algae about 470 million years ago during the early Paleozoic. Hence mosses have a much simpler anatomy than higher land plants such as trees and flowering plants, which in particular means that they have not yet developed a vascular system, i.e an internal transport system for water and nutrients. Since they also lack a complex waterproofing system to prevent absorbed water from evaporating they need a moist environment to grow. Their main habitats are therefore shady and damp places such as woods and edges of streams but they are also found to be resistant to periods of drought and therefore can be found widely spread around the world, from the tropics to tundra regions, from coastal sand dunes up to high mountains.<br />
<br />
The general organization of plant tissue into roots, stem and leaves is found in a much more basic version in mosses. They show a differentiated stem with simple leaves, usually only a single layer of cells thick and lacking veins, that are used to absorb water and nutrients. Instead of roots they have similar threadlike rhizoids [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]]. These have a primary function as mechanical attachment rather than extraction of soil nutrients. Due to not having a vascular system bryophytes are doomed to stay small throughout their life-cycle typically stretching about 1-10 cm.<br />
<br />
However different mosses and vascular plants are because of the early diverge of the evolutionary lineages, they share fundamental genetic and physiological processes. Hence a good approach to studying the complexity of higher land plants is to look at the bryophytes with their much simpler phenotype. Here researchers chose ''Physcomitrella patens'' as a model organism with a genome size of about 450 Mb along 27 chromosomes that is highly similar to other land plants in both exon-intron-structure and codon usage. [[http://www.plantphysiol.org/content/127/4/1430 Schaefer and Zryd, 2001]]<br />
<br />
===Life cycle===<br />
<br />
[[File:TUM13 Physco-lifecycle.png|thumb|right|350px|'''Figure 1:''' ''Physcomitrella'' life cycle]]<br />
Generally land plants show an alternation of generations, the haploid (1n) gametophyte produces sperm and eggs which fuse and transform into the diploid (2n) sporophyte. This then forms haploid spores which become new gametophytes. Besides having no vascular system, bryophytes also differ from higher land plants in the fact that the gametophyte is the dominant phase of their life cycle, whereas in vascular plants the principal generation is the sporophyte.<br />
<br />
The life cycle of ''P. patens'' [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] only takes about 3 months and starts with the spore developing into a filamentous structure, the juvenile, transitory stage of the gametophyte, called protonema, which is composed of two types of cells. The chloronema cells with large and numerous chloroplasts mostly perform photosynthesis and thus supply the photoautotrophic plant with energy while the task of the caulonema cells is fast growth. The adult stage of the gametophyte, called gametophore ("gamete-bearer") has a more complex structure bearing leafs, stem and rhizoids. The transition from juvenile to adult gametophyte is started by initial cells in the protonema filament that differentiate into buds. The budding is therefore a single-cell-event, greatly stimulated by the plant hormone cytokinin, which promotes cell division.<br />
<br />
The sex organs of the moss develop from the tip of the gametophore. ''P. patens'' is monoicous, meaning that male and female organs are produced in one plant. When liquid water is surrounding the tip, flagellate sperm cells can swim from the male sex organ to the female organ and fertilize the egg within. A zygote then develops into the sporophyte, which in turn produces thousands of haploid spores by meiosis. Sporophytes are typically physically attached to and dependent on supply from the dominating gametophyte.<br />
<br />
==Advantages of ''Physcomitrella'' as a model organism==<br />
<br />
===General advantages===<br />
<br />
[[File:TUM13 Bioreactor Cultivation.jpg|thumb|right|350px|'''Figure 2:''' Cultivation of moss in a Bioreactor]]<br />
<br />
*''P. patens'' stands out among the whole plant kingdom as the sole exception where gene targeting is feasible as an easy and fast routine procedure, even with an efficiency similar to ''S. cerevisiae'', due to highly efficient homologous recombination. [[http://www.ncbi.nlm.nih.gov/pubmed/14586556 Reski et al., 2004]] For that reason it is very easy to create knockout mosses by precise mutagenesis following the approach of reverse genetics in order to study the function of genes. Performing functional genomics in higher organisms is very important to understand biological functions of proteins in a multicellular context, e.g. in the context of cell-cell-contacts.<br />
*As mosses mainly are in a haploid stage during their life cycle they are very straight-forward objects for genetics because complex backcrosses to determine changes in the genotype are not necessary<br />
*Moss development starts with a filamentous tissue, the protonema, which is growing by apical cell division and, therefore is perfectly suitable for cell-lineage analysis since development of the plant can be pinpointed to the differentiation of a single cell. [[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] Also the simple life cycle makes ''P. patens'' a very useful item for developmental biology<br />
*''P. patens'' is increasingly used in biotechnology as a study object with implications for crop improvement or human health. Moss bioreactors (see Figure 2) can be used as an alternative to animal cell cultures (e.g. CHO cells) for the easy, inexpensive and safe production of complex biopharmaceuticals [[http://www.sciencedirect.com/science/article/pii/S0958166907000948 Reski and Decker, 2007]] . For example it is a successful tool to produce asialo-EPO, a specific variant of Erythropoetin, which can perform its protective role by inhibiting apoptosis but has lost the potential doping activity. This safe drug is hard to produce in animal cell culture but easy to produce in the moss without impacting its growth or general performance [[http://www.ncbi.nlm.nih.gov/pubmed/22621344 Decker et al., 2012]].<br />
<br />
===Expertbox: Prof. Reski===<br />
[[File:TUM13_expert_Reski.png|thumb|450px|left| '''Figure 3:''' Expert interview with Professor Reski. ]]<br />
When we chose Phytoremediation as our project for this year´s competition, it soon became clear that ''Physcomitrella patens'' is a great chassis that could bring iGEM closer to the real world applications we were looking for. Therefore we contacted Prof. Dr. Reski, who is a recognized expert on ''Physcomitrella patens'' and its biotechnological applications.<br />
<br />
Prof. Dr. Reski liked our idea of introducing his ''Physco'' to iGEM from the very beginning and offered his help wherever we would need it. Members of our team traveled to his lab in Freiburg (350 km) five times in total [https://2013.igem.org/Team:TU-Munich/Results/Moss#3._Transformation_of_Physcomitrella_patens] to fetch plasmids known to be functional in ''Physcomitrealla patens'' or for discussions with him or his co-workers. We could win him over as an advisor for our team, which shows how iGEM brings together universities and scientific groups from many different places.<br />
<br />
===Advantages as a new chassis for iGEM===<br />
*As a plant, ''P. patens'' offers interesting opportunities for application as it is self sustaining, renewable and a natural part of our environment. Therefore it is much easier to implement it into real world scenarios than bacteria or yeast. And although there is the disadvantage of having to wait about 4-6 weeks after transformation until experiments can be done, this is still very short considering the high complexity of the organism. Working with ''P. patens'' can easily be done in the timeframe of the competition by preparing the DNA constructs in bacteria. <br />
*''P. patens'' is a well studied model organism which means that besides having its full genome sequenced in 2006 there are well equipped [http://www.cosmoss.org databases]. Furthermore, there exists an [http://www.moss-stock-center.org International Moss Stock Center (IMSC)] in which many ecotypes, mutants and transgenic strains of ''P. patens'' are stored and accessible to the scientific community. So there is enough knowledge and material to work on for synthetic biologists. <br />
*At the same time the moss offers access to very exciting new physiological processes since it is a much more complex multicellular eukaryotic organism than the chassis already established in iGEM.<br />
*''P. patens'' is an easy plant to work with and requires neither expensive maintenance facilities nor large laboratory space. Most of the basic tools for high precision mutagenesis have been tested on this plant, were found to work and are easily available (see Moss methods below).<br />
<br />
==Moss methods - working with ''Physcomitrella''==<br />
<br />
The techniques used for the cultivation and manipulation of Physcomitrella patens are based on the knowledge of the chair for Plantbiotechnology of Prof. Dr. Reski at Freiburg University, which are available at [http://www.plant-biotech.net Plant-Biotech.net]. <br />
<br />
[[File:TUM13 Bench.png|thumb|right|350px|'''Figure 4:''' Working with cultivated moss on our laboratory bench]]<br />
*<b>Cell culture</b>: ''Physcomitrella patens'' plants can be cultivated either on solidified medium or in liquid medium. Liquid cultures can be kept in Erlenmeyer flasks under constant rotation and light exposure or in bioreactors for large scale production. By regularly (ideally weekly) disrupting the plants mechanically with a mixer (e.g. Ultra-Turrax) a homogenized culture can be achieved.<br />
*<b>Storage</b>: Long-term storage of ''Physcomitrella'' strains can be achieved by cryo-preservation. This procedure ensures a maximum survival rate of the plant by preconditioning and controlled freezing. [[http://www.plant-biotech.net/paper/PlantBiol_2004_Schulte_pageproof.pdf Schulte and Reski, 2004]]<br />
*<b>Targeted knockout</b>: ''P. patens'' is unique among plants in its high efficiency of gene targeting by homologous recombination. To knockout a specific gene a disruption construct has to be generated. This consists of the gene to be silenced with a selection cassette (usually the nptII gene) inserted into its center by suitable restriction sites. The moss is then transformed with this DNA construct which is integrated into the genome by homologous recombination. [[http://www.ncbi.nlm.nih.gov/pubmed/14586556 Reski et al., 2004]]<br />
[[File:TUM13 Transformation.png|thumb|right|350px|'''Figure 5:''' Transformation procedure]]<br />
*<b>Transformation</b>: Transformation of ''P. patens'' requires protoplasts of the plant, which can be obtained by cell wall digesting enzymes. The protoplasts can either be transformed by particle bombardment or via polyethylene glycol (PEG), which is the easiest and most commonly used method. The DNA constructs should be linearized for optimal transformation and contain a selection marker for subsequent screening. After transformation, the protoplasts are cultivated in the dark for 12-16 h followed by 9 days under normal growth conditions during which they regenerate their cell wall. Afterwards they are plated on solidified medium in normal petri dishes and first experiments can be executed. [[http://link.springer.com/article/10.1007/BF00260654 Schaefer et al., 1991]]<br />
*<b>Analysis of transformants</b>: First, the transformed protoplasts are plated on solidified medium containing the antibiotic G418 (Geneticin), to which successfully transformed plants are resistant due to the selection marker nptII which encodes the enzyme neomycin phosphotransferase. Secondly, the transformants are analyzed by a PCR screen using primers derived the selection marker cassette´s sequences [[http://www.plant-biotech.net/paper/PlantMolecularBiologyReporter_2002_Schween.pdf Schween et al., 2002]].<br />
<br />
In '''conclusion''', it naturally takes a bit more time to work with a plant in comparison to bacteria or yeast (see Figure 6), but the advantages definitely outweigh the time factor and if teams start early enough, it is perfectly possible to work with ''P. patens'' as a chassis in an iGEM project!<br />
<br />
[[File:TUM13_timeline.png|thumb|700px| '''Figure 6:''' Comparing work flow for ''E. coli'', ''S. cerevisiae'' and ''P. patens'']]<br />
<br />
This is a brief summary of the most important techniques in working with ''P. patens''. For a full description of these methods please see [https://2013.igem.org/Team:TU-Munich/Notebook/Methods#Physcomitrella_Methods our methods page].<br />
<br />
==BioBricks for protein expression in ''Physcomitrella''==<br />
<br />
[[File:TUM13_plant biobricks.png|thumb|right|300px| '''Figure 7:''' Our transformation vector ]]<br />
To transform the moss, we created an all-purpose backbone into which all our constructs could be integrated easily. Therefore we modified the common iGEM pSB1C3 plasmid with the following plant-specific components:<br />
<br />
* '''pAct5 (Actin 5 promoter from ''Physcomitrella patens''):''' for strong protein expression.<br />
<br />
* '''miniMCS:''' This miniature multiple cloning site contains restriction sites for the enzymes MfeI and SbfI which allows you to insert BioBrick between two other Biobricks (in our case: pAct5 and t35S_npt-cassette) as a result of the compability of MfeI with EcoRI and SbfI with PstI.<br />
<br />
* '''t35S (35S terminator):''' This terminator originates from the 35S transcript of the cauliflower mosaic virus.<br />
<br />
* '''nptII-cassette:''' This selection cassette contains the neomycon phosphotransferase II gene, encoding the enzyme aminoglycoside-3'-phosphotransferase (NPTII), which inactivates a range of aminoglycoside antibiotics such as Kanamycin, Neomycin and Geneticin (G418) by phosphorylation. The latter antibiotic is used as selective agent for transformed ''P. patens'' plants, inhibiting the growth of untransformed plants very effectively. The neomycon phosphotransferase II gene is under control of the NOS promoter and terminator.<br />
<br />
==References:==<br />
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[[http://www.plant-biotech.net/paper/Reski_1998_BotActa-111_1_scan.pdf Reski, 1998]] Reski, R. (1998). Development, Genetics and Molecular Biology of Mosses. ''Bot. Acta'', 111:1-15.<br /><br />
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[[http://www.plantphysiol.org/content/127/4/1430 Schaefer and Zryd, 2001]] Schaefer, D.G. and Zrÿd, J. (2001). The Moss ''Physcomitrella patens'', Now and Then. ''Plant Physiology'', 127(4):1430-1438.<br /><br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/14586556 Reski et al., 2004]] Hohe, A., Egener, T., Lucht, J.M., Holtorf, H., Reinhard, C., Schween, G. and Reski, R. (2004). An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. ''Curr Genet.'', 44(6):339-47.<br /><br />
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[[http://www.sciencedirect.com/science/article/pii/S0958166907000948 Reski and Decker, 2007]] Decker, E.L. and Reski, R. (2007). Moss bioreactors producing improved biopharmaceuticals. ''Current Opinion in Biotechnology'', 18(5):393-398.<br /><br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/22621344 Reski et al., 2012]] Parsons, J., Altmann, F., Arrenberg, C. K., Koprivova, A., Beike, A. K., Stemmer, C., Gorr, G., Reski, R. and Decker, E. L. (2012). Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. ''Plant Biotechnology Journal'', 10:851–861.<br /><br />
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[[http://www.plant-biotech.net/paper/PlantBiol_2004_Schulte_pageproof.pdf Schulte and Reski, 2004]] Schulte, J. and Reski, R. (2004). High throughput Cryopreservation of 140 000 Physcomitrella patens Mutants. ''Plant Biology'', 6:119-127.<br /><br />
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[[http://link.springer.com/article/10.1007/BF00260654 Schaefer et al., 1991]] Schaefer, D., Zryd, J.-P., Knight, C., Cove, D. (1991). Stable transformation of the moss ''Physcomitrella patens''. ''Molecular and General Genetics'', 226:418-424.<br /><br />
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[[http://www.plant-biotech.net/paper/PlantMolecularBiologyReporter_2002_Schween.pdf Schween et al., 2002]] Schween, G., Fleig, S., Reski, R. (2002). High-throughput-PCR screen of 15,000 transgenic Physcomitrella plants. ''Plant Molecular Biology Reporter'', 20:43–47.<br /><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Team/CollaborationsTeam:TU-Munich/Team/Collaborations2013-10-29T02:25:39Z<p>FlorianA: </p>
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==Collaboration with Dundee iGEM team 2013==<br />
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[[File:TUM13_Collaboration_Dundee.png|thumb|right|350px|'''Figure 1:''' Shipment from the Dundee 2013 iGEM team.]]<br />
The [https://2013.igem.org/Team:Dundee Dundee] iGEM team 2013 also works on bioremediation: The toxin microcystin is released into water from lysed cyanobacteria and appears in great amounts during algal blooms. This cyclic peptide toxin covalently binds the protein phosphatase type 1 (PP1) and is thereby toxic for mammals. The idea of the Dundee iGEM team is to express the PP1 protein as an absorber for microcystin. We received their PP1 BioBrick, converted it from <nowiki>RFC 10 to RFC 25</nowiki> and constructed some expression plasmids to transform ''Physcomitrella patens''. Our purpose was applying Dundee's molecular mop in an aquatic, photoautotrophic chassis and thus expand their project's to a wider range. We successfully obtained equipped the moss plants with our synthetic moss receptor and extracellular PP1. '''Please visit our [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PhyscoFilter] results page to read more about this great collaboration.'''<br><br />
We'd also like to congratulate the Dundee iGEM team 2013 for their success at the European Jamboree: '''Well done Scots!'''<br />
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==Collaboration with Glowing Plants==<br />
[[File:TUM13_Glowing_idea.png|thumb|left|300px|'''Figure 2:''' Outline of idea to utilise transgenic moss as ambient light source.]]<br />
[[File:TUM13_Blubber_flasks.gif|thumb|right|700px|'''Figure 3:''' Bubbling flask with our moss. This is how the moss would circulate in the bubble column reactor.]]<br />
We contacted the [http://glowingplant.com/ Glowing Plant] Team which has set itself the goal to develop glowing transgenic ''A. thaliana'' plants using luciferase based on the work of [https://2010.igem.org/Team:Cambridge iGEM Cambridge 2010]. They were quite interested in our idea using transgenic moss plants as light source and we agreed on a meeting on October 25<sup>th</sup>. Ahead of the European Jamboree (October 7 <sup>th</sup> they have also posted our project video on their [http://glowingplant.com/ blog] in order to promote it. We would love to see glowing ''Physcomitrella'' plants in the future.<br />
<br />
Glowingplant performed an extremely successful kickstarter campaign to raise money for their idea of plants which gather energy by photosynthesis and emit it again later by bioluminescence.<br />
<br />
On the 25<sup>th</sup> of October we had the great chance to meet [http://glowingplant.com/team Antony Evans] of the [http://glowingplant.com/ glowingplant team] on his visit to Europe. After having talked about the current status of our projects we had a great knowledge exchange about legal regulations, the public opinion on transgenic plants in Europe and in the US as well as about the potential sizes of the market for transgenic plants for home use. We intend to intensify our collaboration in the fashion of testing their gen cassette used for the luciferase reaction in our moss. It would be interesting to check how the utilized promoters for A. thaliana work in our moss ''P. patens''. However we plan to build a new gene construct using our tested Actin 5 promotor from ''Physcomitrella''.<br />
<br />
Maybe one day our moss will be usable to enlighten our living environment in aerated lava lamps like the one shown in figure 2. Having explored the scientific opportunities of our moss for this purpose glowingplant could become an extremely valuable partner on the way to a commercial product.<br />
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==Collaboration with Paris-Saclay iGEM team 2013==<br />
<br />
[[File:TUM13_Collaboration_Paris.jpg|thumb|right|left|350px|'''Figure 4:''' Screenshot from our Skype conference with the Paris Saclay team.]]<br />
The [https://2013.igem.org/Team:Paris_Saclay Paris_Saclay] iGEM team is working on the detection and degradation of [http://en.wikipedia.org/wiki/Polychlorinated_biphenyl polychlorinated biphenyl] (PCB) in the context of bioremediation. We arranged a Skype-meeting, presented our projects to each other and agreed to '''exchange''' some of our coded '''BioBricks'''. The goal of that exchange was to test the new BioBricks in different chassis.<br />
<br />
==Collaboration with Op.N==<br />
[[File:TUM13_Collaboration_OpN.jpg|thumb|left|350px|'''Figure 5:''' ]]<br />
When we wondered how a moss filter could be implemented, we came across the NY PARALLEL NETWORKS concept which was designed by Op.N (Ali Fard and Ghazal Jafari). [http://op-n.net/filter/office/About-Op-N Op.N's work] considers "critical junction of architecture, landscape, and urban processes". The design and research practice is based in Cambridge and Toronto. Ali Fard and Ghazal Jafari are researchers at Harvard Graduate School of Design. <br />
<br />
We got in touch via E-Mail and found Ali and Ghazal interested in our ideas and '''willing to allow''' us the '''usage''' of their '''image material''' used at the ONE PRIZE competition.<br />
<br />
==Visiting Freiburg iGEM team 2013==<br />
[[File:Muenchen-Freiburg-2013.jpg|thumb|left|350px|'''Figure 6:''' iGEM Teams Freiburg and Munich joint during our first visit]]<br />
[[File:TUM13_Freiburg.JPG|thumb|right|200px|'''Figure 7:''' Our second visit]]<br />
During our [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#Trips_to_Freiburg trip to Freiburg] on August30<sup>th</sup> we met the iGEM team of Freiburg 2013. Along with a guidance through their lab and the Signalhaus they organized a city tour for us. This day ended with a barbecue together with the iGEM team. At this point we would like to thank you for your hospitality!<br />
<br />
== Exchange of urgently needed BioBricks ==<br />
<br />
===LMU Munich iGEM team 2012===<br />
We received the fluorescent proteins GFP, mKate2 and mVenus in RFC 25 from the 2012 Team of LMU Munich.<br />
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===Tuebingen iGEM team 2013===<br />
We sent our pTUM100 vector system ([http://parts.igem.org/Part:BBa_K801000 BBa_K801000]) from the 2012 competition to the iGEM Team of Tuebingen.<br />
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===Uppsala iGEM team 2013===<br />
We provided the iGEM Team Uppsala with CHS consless ([http://parts.igem.org/Part:BBa_K801095 BBa_K801095]), 4CL consless ([http://parts.igem.org/Part:BBa_K801093 BBa_K801093]) and PAL consless ([http://parts.igem.org/Part:BBa_K801091 BBa_K801091]) from the 2012 competition.<br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:24:23Z<p>FlorianA: /* BioAccumulation */</p>
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==Our Project for this summer: Remediation.==<br />
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<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed, the formation of isopeptide bonds were confirmed by pull-down experiments and in reducing SDS-PAGE. Summarizing our results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a chassis.<br />
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==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick Erythromycin Esterase (EreB) which degrades macrolide antibiotics. On the other hand we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular portion of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life was estimated in river water. All data was fitted in our enzyme kinetic modeling. The aim was to analyze these data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football field would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
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==BioAccumulation==<br />
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Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
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==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a close contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein which then is liberated and can degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==The iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years and we also invested some effort to advance the iGEM community. We programed a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and does alignments with various data banks. In the end all collected information for a BioBrick is collected in a standardized table which can easily be integrated into the part description of a BioBricks. We submitted a RFC for this [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Beside this software toll we have written tutorials on wiki programming, creation of animated gifs of protein structures and the usage of Arduino microcontrollers for iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:23:40Z<p>FlorianA: /* Our Project for this summer: Remediation. */</p>
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==Our Project for this summer: Remediation.==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 0px 0px 20px 20px;background-color: white; float: right;" src="http://player.vimeo.com/video/76195786" width="400" height="240" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<p>During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.</p><br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed, the formation of isopeptide bonds were confirmed by pull-down experiments and in reducing SDS-PAGE. Summarizing our results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick Erythromycin Esterase (EreB) which degrades macrolide antibiotics. On the other hand we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular portion of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life was estimated in river water. All data was fitted in our enzyme kinetic modeling. The aim was to analyze these data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football field would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;float: right;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html> <br><br />
Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a close contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein which then is liberated and can degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==The iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years and we also invested some effort to advance the iGEM community. We programed a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and does alignments with various data banks. In the end all collected information for a BioBrick is collected in a standardized table which can easily be integrated into the part description of a BioBricks. We submitted a RFC for this [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Beside this software toll we have written tutorials on wiki programming, creation of animated gifs of protein structures and the usage of Arduino microcontrollers for iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:15:35Z<p>FlorianA: /* BioAccumulation */</p>
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==Our Project for this summer: Remediation.==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white; float: right;" src="http://player.vimeo.com/video/76195786" width="400" height="240" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html> <br><br />
During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.<br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed, the formation of isopeptide bonds were confirmed by pull-down experiments and in reducing SDS-PAGE. Summarizing our results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick Erythromycin Esterase (EreB) which degrades macrolide antibiotics. On the other hand we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular portion of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life was estimated in river water. All data was fitted in our enzyme kinetic modeling. The aim was to analyze these data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football field would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;float: right;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html> <br><br />
Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a close contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein which then is liberated and can degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==The iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years and we also invested some effort to advance the iGEM community. We programed a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and does alignments with various data banks. In the end all collected information for a BioBrick is collected in a standardized table which can easily be integrated into the part description of a BioBricks. We submitted a RFC for this [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Beside this software toll we have written tutorials on wiki programming, creation of animated gifs of protein structures and the usage of Arduino microcontrollers for iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Results/SummaryTeam:TU-Munich/Results/Summary2013-10-29T02:14:56Z<p>FlorianA: /* Our Project for this summer: Remediation. */</p>
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==Our Project for this summer: Remediation.==<br />
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During this summer we wanted to work on an iGEM project, which has the potential to become a real world application, since we believe that it is an important step for Synthetic Biology to provide alternative solutions for global problems. For this reason we focused on Bioremediation: The use of organisms to remove emissions caused by humans and bring the environment back to its natural state. As water is a resource which is absolutely essential for all living organisms, we decided to focus on the pollution of aquatic ecosystems.<br />
<br />
==Choice of the appropriate chassis for a water filter==<br />
Remediation is not new to iGEM, in fact it is a topic the iGEM community has worked on for nearly 10 years now. Therefore a set of promising BioBricks were already available in the Parts Registry, which we wanted to use in order to increase the knowledge on these effector proteins. Having found suitable effector proteins, we discussed about the most suitable chassis for our application. Most of the previous projects on Bioremediation were based on ''E. coli'', whereas we decided to use a plant instead. Photosynthesis carried out by the plants will allow the water filter to maintain and renew itself without the addition of any nutrients. We considered algae such as ''Chlamydomonas reinhardtii'', Bryophytes such as ''Physcomitrella patens'' and higher plants like ''Arabidopsis thaliana''. In the end ''Physcomitrella patens'' was the chassis of choice as it already grows in a filter-like structure and can be cultivated in terrestric as well as in aquatic conditions. Additionally it is a well established organism in biotechnology. Working with ''Physcomitrella patens'' is not easy considering the 1-2 months it takes from the transformation process to the experiments with stable transfected plants and the doubling times of 3-6 days. As nobody at the TU Munich works with the moss ''Physcomitrella patens'', we looked for an expert and found Prof. Reski who occupies a professorship at Freiburg University. We were very happy to gain him as an advisor during our project. For the use of ''Physcomitrella patens'' in iGEM we created a strong constitutive promoter ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306]), a plant terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159307 BBa_K1159307]) and an antibiotic selection marker ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159308 BBa_K1159308]), which were all used to transform and select 21 different transgenic moss lines.<br />
<br />
==Localization of effector proteins==<br />
[[File:Localization_general.jpg|thumb|right|500px|'''Figure 1:''' Cytosolic protein expression and our modularized receptor for ''Physcomitrella'' work as expected.]]<br />
The actual remediation of pollutants is accomplished by effector proteins which function with quite different mechanisms. Thus it was important to enable the localization of effector proteins at different cellular sites. Cytosolic effector proteins are easily expressed, whereas for secretion a signal peptide BioBrick is cloned ahead of the effector protein. Several receptor signal peptides from ''Physcomitrella patens'' and other organisms were analyzed by using [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics tools]. The signal peptide of the Somatic Embryogenesis Receptor-like Kinase (SERK) from ''Physcomitrella patens''and the IgK signal peptide from ''Mus musculus'', which is described in literature to function in ''Physcomitrella'', were chosen. The secretion of a newly introduced luciferase with both of these signal peptides was investigated for 8 clones each. No detectable secretion for the IgKappa but a high secretion rate for the SERK signal peptide was shown. Successful secretion could be achieved using the SERK signal peptide. Because the secreted effector proteins are not attached to the moss cell, they diffuse into the water, which is suboptimal, for example if you want to remove pollutants by simply binding them. Therefore we designed a modular receptor for ''Physcomitrella'', which can carry effector proteins at the outer side of the cell membrane. For this purpose [https://2013.igem.org/Team:TU-Munich/Modeling/Protein_Predictions bioinformatics methods] were used again and the SERK transmembrane domain was chosen to be the best one. A receptor composed of (1) the SERK signal peptide, (2) an extracellularly located effector protein, (3) a linker with a Strep-tag II and a TEV protease cleavage site, (4) the SERK transmembrane domain, (5) a short linker domain and (6) a green fluorescent protein were assembled using the RFC[25] standard. This highly modular receptor was successfully transformed into ''Physcomitrella patens'' and stable cell lines were selected. These stable cell lines were used for experiments. The localization of the membrane-bound GFP could be detected clearly on the surface of the moss cells (see figure 1), whereas expressing GFP cytosolically in the moss, showed a uniform fluorescence over the whole cell. Further we incubated the moss cells with recombinant TEV protease, which diffused through the cell wall, cleaved the TEV site within the extracellular domain of the receptor and liberated the NanoLuc luciferase. The luciferase assay of the supernatant at the beginning of this incubation and after 16 hours showed a dramatic increase in luminescence, which is an evidence that our modular receptor is located in the membrane and - even better - in the right orientation, exposed to the extracellular space. Beyond the possibility to locate an effector protein in the extracellular space, we thought about further applications and found the SypCatcher-SpyTag System to be a perfect tool for our needs [[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]]. In this system a peptide bond is formed between the side chains of two protein domains in an efficient manner. With this system it is not necessary to fuse effector proteins to a specific terminus of the receptor any more, it gets even possible to immobilize effector proteins which are only active as multimeric proteins and it would also be feasible to express a single receptor carrying a SpyCatcher domain at the outer side of the membrane which subsequently binds a set of different effector proteins which are secreted and get immobilized afterwards. Constructs were created with a His-tagged SypCatcher, a SypTag with a N-terminal or C-terminal SpyTag or with protein domains on both termini of the SpyTag. These constructs were produced recombinantly in ''E. coli''. Afterwards the proteins were purified. Protein coupling experiments were performed, the formation of isopeptide bonds were confirmed by pull-down experiments and in reducing SDS-PAGE. Summarizing our results, all our intended localizations in ''Physcomitrella patens'' worked, empowering the iGEM community to work creatively with ''Phycomitrella patens'' as a chassis.<br />
<br />
==BioDegradation==<br />
[[File:TUM13_EreB_LCMS.png|thumb|right|350px|'''Figure 2''': Degradation of erythromycin by recombinant protein and our PhyscoFilter [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Erythromycin read more].]]<br />
Under the headline [https://2013.igem.org/Team:TU-Munich/Project/Biodegradation BioDegradation] we investigated effector proteins which degrade pollutants by enzymatic catalysis. For this purpose we introduced the new enzyme BioBrick Erythromycin Esterase (EreB) which degrades macrolide antibiotics. On the other hand we used well established BioBricks for BioDegradation. We improved the Laccase from Bacillus pumilus by converting it to RFC[25] in order to be able to integrate it into the extracellular portion of our receptor. Both enzymes were produced as recombinant proteins and were purified. Enzymatic characterization was carried out concerning substrate dependency, salt tolerance and pH dependency. For the laccase additionally the temperature dependency and the half-life was estimated in river water. All data was fitted in our enzyme kinetic modeling. The aim was to analyze these data and to provide a solid base for our filter calculator. This calculator uses all data produced in our enzyme characterization to extrapolate for the use of transgenic PhyscoFilter in waste water treatment plants or rivers. We assumed the secretory production of laccase by our moss as the laccase degrades a wide variety of important pollutants such as the pain killer diclofenac, the oral contraceptive ethinylestradiol or iodined x-ray contrast media which are all present in nature and are hardly degradable by conventional methods. From the [https://2013.igem.org/Team:TU-Munich/Modeling/Filter modeling with this calculator] we learned that factor such as the degree of pollution of a river, the average temperature in a specific country, the enzyme half-life as well as the actual amount of secreted protein play an important role for the efficacy of our PhyscoFilter. Generally the results show that approximately an area of 20 football field would be required to produce enough laccase to reduce the contamination of a river with the mentioned pharmaceutical compounds. Beside this result we also produced several different stable transgenic moss lines for our BioDegradation module and could show that our cytoplasmatically expressed Erythromycin Esterase B enables our moss to degrade the macrolide antibiotic Erythromycin which is normally only degraded poorly. This experiment was measured with mass spectrometry coupled to liquid chromatography (LC-MS) and worked for the recombinant protein as well as for the transgenic plant giving the proof of principle for ''Physcomirella'' as a bioremediation organism.<br />
<br />
==BioAccumulation==<br />
<html><center><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/77974681" width="400" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></center></html> <br><br />
Beside the enzymatic degradation of pollutants we found different methods to bind pollutants to our moss filter which we called [https://2013.igem.org/Team:TU-Munich/Project/Bioaccumulation BioAccumulation]. The most obvious idea was to use binding proteins which are developed for human therapy such as antibodies for example. To investigate this idea we used an alternative binding protein (Anticalin) engineered to bind fluorescein as it has a very high affinity, a small size and a robust fold [[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]. Beside this engineered binding protein we also found the idea of [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 Dundee iGEM 2013] quite interesting to use the protein which is affected in the toxicity mechanism of microcystein and to use it as a binding partner which then absorbs the pollutant from the water. Thus we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration%20with%20Dundee%20iGEM%20team%202013 contacted Dundee iGEM], told them about our idea of an collaboration, got their BioBrick sent, converted it to RFC[25], assembled it into our modular receptor and finally transformed and selected stable transfected moss lines which we characterized finally. Basically the limitation of BioAccumulation applications is that they only can bind one pollutant per binding protein and thus an extremely high number of binding proteins is required to achieve a reduction of environmental pollutants. We transformed and selected transgenic moss lines with all three effector proteins and checked the cellular localization of these proteins using light microscopy. For the moss lines with a receptor harboring an Anticalin which binds fluorescein a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Fluorescein membrane bound localization could be confirmed] whereas the moss lines with a receptor carrying the protein phosphatase 1 (PP1) from our collaboration partner Dundee showed a [https://2013.igem.org/Team:TU-Munich/Results/GM-Moss#The_PhyscoFilter_for_Microcystin localization in cytosolic vesicles] and not on the membrane. This is in good agreement with the result presented by Dundee that the SEC-pathway secretion is not working for this BioBrick. This might be because of surface exposed cystein residues which tend to aggregation in the oxidizing milieu, therefore it would be necessary to perform protein engineering to exchange these cystein residues for other amino acid residues in order to increase the stability of this protein.<br />
<br />
==Kill-Switch==<br />
Safety is one of the most important issues in synthetic biology and we therefore implemented a kill switch into our project. For us it was important to use a trigger which is ubiquitously present in the environment and no action of humans is required. We therefore developed a light-triggered kill switch. With this system the transgenic moss could be cultivated under blue filter foil. As long as the moss grows under this blue foil no red light reaches the moss and the moss stays alive. As soon as the moss escapes from this protected environment the red light is present and the kill switch becomes triggered. The system was modularized into a sensor module and a suicide module. The sensor domain consists of a splitted TEV protease which is attached to either PhyB or PIF. The later two proteins dimerized when red light is present and therefore lead to the reconstitution of the TEV protease. The suicide module consists of nuclease which is localized at the cellular membrane by a linker which contains a nuclear localization signal (NLS) and a TEV cleavage site. As soon as the sensor module is reconstituted by red light, the TEV protease cleaves its cleavage site inside the suicide module. Thereby the nuclease is liberated, becomes transported to the nucleus because of the nuclear localization signal and fragments the genome. The choice to use a nuclease instead of siRNA for example was driven by our modeling in which we found the siRNA suicide module to be less effective as there is a negative feedback loop which avoids the efficient killing of moss cells. <br />
We have transformed moss cells with this kill switch and have protected the resulting cells by the blue foil mentioned before. When we opened the blue foil after the selection process, all moss cells were dead. This can be explained by a drastically reduced transformation efficacy as the kill switch DNA was >10 kDa or by the fact that the kill switch is reliably killing the cells even without a trigger. In order to test the sensor module in vitro we have produced the two fusion proteins in vitro as recombinant proteins and have attempted to purify them which was not successful as the proteins are most probably not stable in vitro. Although we only had a single shot to test our kill switch in ''Physcomitrella'' we have discussed by far more about this system compared to the other parts of our project that worked very well in first experiments. During these discussions on our kill switch we have learned a lot about this system and we described these findings in order to help subsequent iGEM teams which are aiming to design a comparable kill switch.<br />
<br />
==Implementation==<br />
[[File:TUM13_RenderingMIT.jpg|aft.png|thumb|right|400px|'''Figure 3''': Remediation rafts in front of the MIT]]<br />
Projects in iGEM must not stop at the lab door and therefore it is immensely important to think about technical solutions to implement the transgenic organisms in order to show highest efficacy. For this reason we convinced experts like Prof. Dr. Posten to join our [https://2013.igem.org/Team:TU-Munich/HumanPractice/Interviews#Expert_Counsel:_An_overview advisery board] and have evaluated different cultivation methods for moss such as closed tube reactors, open pond reactors and floating remediation rafts. We came to the conclusion that in the case of immobilized effector proteins an open pond or closed tube reactor will be the superior technology as the degradation requires a close contact between the moss and a the pollutant to degrade. As a second possibility we evaluated the secretion of effector proteins such as laccase, which would then be implemented best on floating remediation rafts which are cheap to produce, mobile and could also be applied in third world countries. PhyscoFilter moss could be grown on these rafts and would secrete recombinant protein which then is liberated and can degrade pollutants in the environment. For all these cultivation methods we built model reactors, tried the cultivation of moss within them and tested the flow characteristics of the systems. For the triangular remediation raft we constructed a life-size prototype which costed only US$ 70. Additionally we developed a measurement device based on an [https://2013.igem.org/Team:TU-Munich/Results/How_To#Setting_up_a_basic_Arduino_measuring_device Arduino microcontroller] which measures environmental parameters, sends the data via WiFi to a webserver from where the actual data can be monitored with any smart phone or computer at any place in the world. To get an idea how such remediation rafts could look like on our rivers, we [https://2013.igem.org/Team:TU-Munich/Team/Collaborations#Collaboration_with_Op.N talked to architects] and also rendered an 3D-CAD-model in front of the MIT (see figure 3).<br />
<br />
==The iGEM Community==<br />
It is really amazing to see how the iGEM community advanced over the last years and we also invested some effort to advance the iGEM community. We programed a software tool which translates protein coding BioBricks in the registry to amino acid sequences, calculates various parameters and does alignments with various data banks. In the end all collected information for a BioBrick is collected in a standardized table which can easily be integrated into the part description of a BioBricks. We submitted a RFC for this [https://2013.igem.org/Team:TU-Munich/Results/Software AutoAnnotator] which obtained the number RFC[96].<br><br />
Beside this software toll we have written tutorials on wiki programming, creation of animated gifs of protein structures and the usage of Arduino microcontrollers for iGEM projects.<br />
<br />
==References:==<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]] Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Jan;7(1):73-86<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012]] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. ''Proc Natl Acad Sci U S A''. 20;109(12)<br><br />
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{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Modeling/Kill_SwitchTeam:TU-Munich/Modeling/Kill Switch2013-10-29T02:03:40Z<p>FlorianA: </p>
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==Kill Switch Modeling==<br />
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====Purpose====<br />
<br />
The idea of our [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] is to kill off our moss, as soon as it leaves the filter system. For this purpose two methods were proposed:<br />
#'''siRNA''' method: When some trigger is activated, siRNA is expressed inhibiting the expression of a vital gene<br />
#'''nuclease''' method: When some trigger is activated, a nuclease is released destroying the DNA of the cell<br />
<br />
To decide between these two methods we modelled the vitality V of the cell (a number between 0 and 1, so a perfectly functional cell has V=1, a dead cell V=0) and depending on the tested method the concentration of siRNA R and nuclease N as appropriate. Both concentrations are normalized to the unit interval [0,1].<br />
<br />
==siRNA Model==<br />
<br />
====Governing equations====<br />
[[File:TUM13_siRNA_graph.png|thumb|right|400px| '''Figure 1:''' Example solution to our siRNA model with parameters k<sub>1</sub>=1, k<sub>2</sub>=2, k<sub>3</sub>=2, k<sub>4</sub>=1.]]<br />
<br />
We determined the governing equations of this model to be the following:[[File:TUM13_siRNA_formula.png|center]]<br />
<br />
with initial conditions V(0) = 1 and R(0) = 0, where at the time t=0 the trigger is activated.<br />
<br />
Figure 1, created by our MatLab script [[#siRNA_model_script|siRNA model]] shown below, on the right shows a solution to this system for some example parameters. In this case the vitality of the cell decreases to somewhere around 0.3 while at the same time after an initial peak the siRNA concentration also drops upon which the vitality settles in at about 0.4.<br />
<br />
So it appears, that the siRNA approach does not achieve the required death of the cells. In the following it will be shown, that this is the case for all parameter values.<br />
<br />
====Calculation of stable points and analysis====<br />
<br />
The steady points V* and R* of this system have to satisfy [[File:TUM13_siRNA_stable_satisfy.png|center]]<br />
<br />
Defining [[File:TUM13_siRNA_alpha_beta_def.png]] we get the following quadratic equation for the steady point of V [[File:TUM13_siRNA_stable_quadratic.png|center]]<br />
<br />
<br />
'''If &alpha; = 1''', the unique steady point is [[File:TUM13_siRNA_alphaIS1_stable.png]].<br />
<br />
To analyze the stability of these the eigenvalues of the Hessian matrix H [[File:TUM13_siRNA_alphaIS1_Hessian.png|center]]<br />
<br />
must be computed. The eigenvalues are [[File:TUM13_siRNA_alphaIS1_EV.png|center]]<br />
<br />
<br />
These are both negative, so this is a '''stable point''', i.e. an attractor.<br />
<br />
<br />
'''If &alpha; ≠ 1''', the steady points are [[File:TUM13_siRNA_stable_points.png|center]]<br />
<br />
<br />
Now only one of these is in the sensible range, because<br />
*for &alpha; > 1: [[File:TUM13_siRNA_alphaGT1_lowerbound.png]]<br />
*for &alpha; < 1: [[File:TUM13_siRNA_alphaLT1_lowerbound.png]]<br />
<br />
So there is only one steady point in this range, namely:<br />
[[File:TUM13_siRNA_stable_realistic_point.png|center]]<br />
<br />
<br />
By expanding the fraction by [[File:TUM13_siRNA_expandby.png]] we can rewrite V* as<br />
[[File:TUM13_siRNA_V_rewritten.png|center]]<br />
<br />
<br />
So using the results from above, we get: [[File:TUM13_siRNA_V_in01.png]]<br />
<br />
Now look at the eigenvalues of the Hessian matrix to analyze the stability [[File:TUM13_siRNA_Hessian.png|center]]<br />
<br />
<br />
Defining [[File:TUM13_siRNA_EigVals_b.png]], the eigenvalues are given by<br />
[[File:TUM13_siRNA_EigVals.png|center]]<br />
<br />
<br />
So [[File:TUM13_siRNA_END.png]], which means that this is a '''stable attractor'''.<br />
<br />
<br />
====Result of our model====<br />
<br />
So for the '''siRNA''' there '''always is a stable point''' for the vitality '''between (but excluding) 0 and 1''', so the moss is '''not killed-off''' completely, just impeded in its growth.<br />
<br />
<br />
====Interpretation====<br />
<br />
This result makes intuitive sense, because as the function of the cell is repressed the cell produces less of the inhibiting siRNA, which leads to a regeneration of the cell. Eventually a steady state at a lower vitality is reached, where the vitality stays constant.<br />
<br />
==== Stochastic Model ====<br />
<br />
[[File:TUM13_SiRNA_stoch.png|thumb|right|400px| '''Figure 2''': Averaging over 1000 realisations of the Gillespie algorithm. Solid line indicates the mean, dashed lines indicate the 1-&sigma; interval.]]<br />
<br />
As number of siRNA in a cell can be very small, we cannot always assume that there is an sufficient amount of RNA such we can rule out stochastic effect. To deal with this problem we set up an analogous stochastic model constisting of a Markov Jump Process of the two species with a total of 4 reactions.<br />
<br />
[[File:TUM13_SiRNA_stoch_system.png|center|400px]]<br />
<br />
<br />
The state space [0,1] was discretised to [1,2,3,...,100] for both species R and N while the reaction rates were kept the same.<br />
<br />
To simulate the model we used and implementation of the Gillespie Algorithm with 1000 realisations and then averaged over all realisations.<br />
<br />
With this implementation the qualitative behaviour of the system remains unchanged, while the quantitative changes which is due to the discretization.<br />
<br />
==Nuclease Model==<br />
<br />
====Governing equations====<br />
[[File:TUM13_nuc_graph.png|thumb|right|400px| '''Figure 3:''' Example solution to our nuclease model with parameters p<sub>1</sub>=1, p<sub>3</sub>=0.5, p<sub>4</sub>=0.005.]]<br />
We determined the governing equations of this model to be the following:[[File:TUM13_nuc_formula.png|center]]<br />
<br />
with initial conditions V(0) = 1 and N(0) = 0, where at the time t=0 the trigger is activated.<br />
<br />
Figure 2, created by our MatLab script [[#nuclease_model_script|nuclease model]] shown below, on the left shows a solution to this system for some example parameters. It clearly shows that in this case the vitality of the cell decreases to 0 and remains there, i.e. that the cell has died.<br />
<br />
In the following we will verify, that this is the case for all parameter values.<br />
<br />
====Calculation of stable points and analysis====<br />
<br />
Any steady state given by V* and N* of this system must to satisfy [[File:TUM13_nuc_stable_satisfy.png|center]]<br />
<br />
<br />
The Hessian matrix of this system for the steady point is easy to solve [[File:TUM13_nuc_hessian_ev.png|center]]<br />
<br />
<br />
The eigenvector corresponding to the zero eigenvalue is [[File:TUM13_nuc_eigenvec.png]]. It is obvious from the equations that any disturbance away from the steady state along this vector will decay back to the steady state, '''so this is a stable attractor'''.<br />
<br />
====Results of our model====<br />
<br />
The '''nuclease''' always reduces the vitality of the moss to 0, i.e. '''kills it off completely'''.<br />
<br />
<br />
====Interpretation====<br />
<br />
Again this result is very intuitive, since the '''moss cannot regenerate''' from the destruction of its genome.<br />
<br />
===Conclusions===<br />
<br />
For a functional kill-switch it is necessary, that the cells are actually killed and not just live on with reduced vitality. So based on our modeling results the siRNA approach is not satisfactory, while the '''nuclease satisfies the requirement'''. '''As a result the team pursued the nuclease approach leading to our final kill-switch.'''<br />
<br />
<br />
==MatLab Scripts==<br />
<br />
====siRNA model script====<br />
<br />
<nowiki>% f = @(R,V,k) [k(3)* V - k(4) * R , -k(1) * R * V + k(2) *(V -1)* (R -1)];<br />
g = @(k,y) [k(3)* y(2)- k(4)*y(1); -k(1)*y(1)*y(2)+k(2)*(y(2)-1)*(y(1)-1)];<br />
<br />
%k= [ k1, k2, k3, k4 ]; %insert the appropriate reaction rate constant<br />
k = [ 1, 2, 2, 1];<br />
<br />
[TOUT, YOUT] = ode45(@(t,y) g(k,y) ,[0,10],[0;1]);<br />
<br />
plot(TOUT, YOUT(:,1), 'markersize', 15, 'linewidth', 5)<br />
hold on; plot(TOUT, YOUT(:,2), 'r', 'markersize', 15, 'linewidth', 5); hold off<br />
<br />
legend('siRNA concentration','Vitality');<br />
xlabel('time');<br />
set(gca,'FontSize',24);<br />
set(gcf,'position', [100 100 600 600]);<br />
<br />
axis square<br />
set(gcf,'Color','w');<br />
export_fig siRNA_pic.png<br />
</nowiki><br />
<br />
==== Stochastic siRNA script ====<br />
<br />
<nowiki>%% 1 States<br />
% [R,V]<br />
x0 = [0,100];<br />
<br />
%% 2 Reactions<br />
% stoichiometric matrix<br />
S = zeros(4,2);<br />
<br />
%% 2.1 increase siRNA<br />
% R -> R + 1<br />
% rate k3 * V<br />
S(1,:) = [1,0];<br />
<br />
%% 2.2 degrade siRNA<br />
% R -> R - 1<br />
% rate k4 * R<br />
S(2,:) = [-1,0];<br />
<br />
%% 2.3 increase vitality<br />
% V -> V + 1<br />
% rate k2 * ( V - 1 ) * ( R - 1 )<br />
S(3,:) = [0,1];<br />
<br />
%% 2.4 decrease vitality<br />
% V -> V - 1<br />
% rate k1 * R * V<br />
S(4,:) = [0,-1];<br />
<br />
<br />
param = [ 1, 2, 1, 2];<br />
<br />
% rates<br />
acell = { @(t,x,k) k(3) * x(2)/100,<br />
@(t,x,k) k(4) * x(1)/100,<br />
@(t,x,k) k(2) * ( x(2) - 100 )/100 * ( x(1) - 100 )/100,<br />
@(t,x,k) k(1) * x(1) * x(2)/100};<br />
<br />
a = @(t,x,k) [feval(acell{1},t,x,k),feval(acell{2},t,x,k),feval(acell{3},t,x,k),feval(acell{4},t,x,k)];<br />
<br />
% time vector<br />
<br />
N_time = 100;<br />
<br />
tt = linspace(0,100,N_time);<br />
<br />
% number of runs<br />
N_repeat = 1000;<br />
<br />
% output<br />
X_runs = zeros(N_repeat,N_time,length(x0));<br />
<br />
<br />
%plotting<br />
figure(1)<br />
clf<br />
hold on<br />
<br />
for j = 1:N_repeat <br />
[X_runs(j,:,:)]=SSA(x0,S,a,tt,param);<br />
end<br />
<br />
figure<br />
hold on<br />
plot(tt,mean(X_runs(:,:,1)),'b')<br />
plot(tt,mean(X_runs(:,:,2)),'r')<br />
legend('siRNA','Vitality')<br />
for k = 1:length(x0)<br />
switch k<br />
case 1<br />
plot(tt,mean(X_runs(:,:,k)),'b')<br />
plot(tt,mean(X_runs(:,:,k))+sqrt(var(X_runs(:,:,k))),'--b')<br />
plot(tt,mean(X_runs(:,:,k))-sqrt(var(X_runs(:,:,k))),'--b')<br />
case 2<br />
plot(tt,mean(X_runs(:,:,k)),'r')<br />
plot(tt,mean(X_runs(:,:,k))+sqrt(var(X_runs(:,:,k))),'--r')<br />
plot(tt,mean(X_runs(:,:,k))-sqrt(var(X_runs(:,:,k))),'--r')<br />
end<br />
end<br />
<br />
find(X_runs(:,:,2)==0,1,'first')<br />
set(gcf,'Color','w')<br />
axis square<br />
export_fig siRNA_stochastic.png<br />
</nowiki><br />
<br />
====Gillespie====<br />
<br />
<nowiki>function [ xx, XX, TT ] = SSA( x0, S, a, tt , param)<br />
%SSA Summary of this function goes here<br />
% Detailed explanation goes here<br />
<br />
% initialise<br />
TT(1) = 0;<br />
XX(1,:) = x0;<br />
<br />
% step<br />
i = 1;<br />
while (TT(end)<tt(end));<br />
% increment step<br />
i = i + 1;<br />
<br />
%compute rates<br />
at = a(TT(i-1),XX(i-1,:),param);<br />
a0 = sum(at);<br />
<br />
for k = 1 : length(at)<br />
aj(k) = sum(at(1:k)/a0);<br />
end<br />
<br />
%sample time<br />
TT(i) = TT(i-1) + expinv(rand,1/a0);<br />
<br />
% select reaction that happens<br />
j = find(aj>rand,1,'first');<br />
<br />
% add reaction<br />
XX(i,:) = XX(i-1,:) + S(j,:);<br />
end<br />
<br />
xx = zeros(length(tt),length(x0));<br />
for j = 1:length(tt)<br />
xx(j,:) = XX(find(TT<=tt(j),1,'last'),:);<br />
end<br />
<br />
end<br />
</nowiki><br />
<br />
====Nuclease model script====<br />
<br />
<nowiki>% f = @(N,V,k) [p(3)* V - p(4) * N , -p(1) * N * V ];<br />
g = @(p,y) [p(3)* y(2)- p(4)*y(1); -p(1)*y(1)*y(2)];<br />
<br />
%p= [ p1, p2, p3, p4 ]; %insert the appropriate reaction rate constant<br />
p = [ 1, NaN, 1, 0.5 ];<br />
<br />
[TOUT, YOUT] = ode45(@(t,y) g(p,y) ,[0,20],[0;1]);<br />
<br />
plot(TOUT, YOUT(:,1), 'markersize', 15, 'linewidth', 5)<br />
hold on; plot(TOUT, YOUT(:,2), 'r', 'markersize', 15, 'linewidth', 5); hold off<br />
<br />
legend('Nuclease concentration','Vitality')<br />
xlabel('time');<br />
set(gca,'FontSize',24);<br />
set(gcf,'position', [100 100 600 600]);<br />
<br />
axis square<br />
set(gcf,'Color','w')<br />
export_fig nuc_pic.png</nowiki><br />
<br />
</div><br />
<!-- Ende des Inhalts --><br />
</div><br />
</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianAhttp://2013.igem.org/Team:TU-Munich/Modeling/Kill_SwitchTeam:TU-Munich/Modeling/Kill Switch2013-10-29T02:02:58Z<p>FlorianA: </p>
<hr />
<div>{{Team:TU-Munich/TUM13_Menu}}<br />
{{Team:TU-Munich/TUM13_Style}}<br />
<br />
<div id="wikicontent-container"><br />
<div id="wikicontent"><br />
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<div><br />
==Kill Switch Modeling==<br />
<br />
====Purpose====<br />
<br />
The idea of our [https://2013.igem.org/Team:TU-Munich/Project/Killswitch kill switch] is to kill off our moss, as soon as it leaves the filter system. For this purpose two methods were proposed:<br />
#'''siRNA''' method: When some trigger is activated, siRNA is expressed inhibiting the expression of a vital gene<br />
#'''nuclease''' method: When some trigger is activated, a nuclease is released destroying the DNA of the cell<br />
<br />
To decide between these two methods we modelled the vitality V of the cell (a number between 0 and 1, so a perfectly functional cell has V=1, a dead cell V=0) and depending on the tested method the concentration of siRNA R and nuclease N as appropriate. Both concentrations are normalized to the unit interval [0,1].<br />
<br />
==siRNA Model==<br />
<br />
====Governing equations====<br />
[[File:TUM13_siRNA_graph.png|thumb|right|400px| '''Figure 1:''' Example solution to our siRNA model with parameters k<sub>1</sub>=1, k<sub>2</sub>=2, k<sub>3</sub>=2, k<sub>4</sub>=1.]]<br />
<br />
We determined the governing equations of this model to be the following:[[File:TUM13_siRNA_formula.png|center]]<br />
<br />
with initial conditions V(0) = 1 and R(0) = 0, where at the time t=0 the trigger is activated.<br />
<br />
Figure 1, created by our MatLab script [[#siRNA_model_script|siRNA model]] shown below, on the right shows a solution to this system for some example parameters. In this case the vitality of the cell decreases to somewhere around 0.3 while at the same time after an initial peak the siRNA concentration also drops upon which the vitality settles in at about 0.4.<br />
<br />
So it appears, that the siRNA approach does not achieve the required death of the cells. In the following it will be shown, that this is the case for all parameter values.<br />
<br />
====Calculation of stable points and analysis====<br />
<br />
The steady points V* and R* of this system have to satisfy [[File:TUM13_siRNA_stable_satisfy.png|center]]<br />
<br />
Defining [[File:TUM13_siRNA_alpha_beta_def.png]] we get the following quadratic equation for the steady point of V [[File:TUM13_siRNA_stable_quadratic.png|center]]<br />
<br />
<br />
'''If &alpha; = 1''', the unique steady point is [[File:TUM13_siRNA_alphaIS1_stable.png]].<br />
<br />
To analyze the stability of these the eigenvalues of the Hessian matrix H [[File:TUM13_siRNA_alphaIS1_Hessian.png|center]]<br />
<br />
must be computed. The eigenvalues are [[File:TUM13_siRNA_alphaIS1_EV.png|center]]<br />
<br />
<br />
These are both negative, so this is a '''stable point''', i.e. an attractor.<br />
<br />
<br />
'''If &alpha; ≠ 1''', the steady points are [[File:TUM13_siRNA_stable_points.png|center]]<br />
<br />
<br />
Now only one of these is in the sensible range, because<br />
*for &alpha; > 1: [[File:TUM13_siRNA_alphaGT1_lowerbound.png]]<br />
*for &alpha; < 1: [[File:TUM13_siRNA_alphaLT1_lowerbound.png]]<br />
<br />
So there is only one steady point in this range, namely:<br />
[[File:TUM13_siRNA_stable_realistic_point.png|center]]<br />
<br />
<br />
By expanding the fraction by [[File:TUM13_siRNA_expandby.png]] we can rewrite V* as<br />
[[File:TUM13_siRNA_V_rewritten.png|center]]<br />
<br />
<br />
So using the results from above, we get: [[File:TUM13_siRNA_V_in01.png]]<br />
<br />
Now look at the eigenvalues of the Hessian matrix to analyze the stability [[File:TUM13_siRNA_Hessian.png|center]]<br />
<br />
<br />
Defining [[File:TUM13_siRNA_EigVals_b.png]], the eigenvalues are given by<br />
[[File:TUM13_siRNA_EigVals.png|center]]<br />
<br />
<br />
So [[File:TUM13_siRNA_END.png]], which means that this is a '''stable attractor'''.<br />
<br />
<br />
====Result of our model====<br />
<br />
So for the '''siRNA''' there '''always is a stable point''' for the vitality '''between (but excluding) 0 and 1''', so the moss is '''not killed-off''' completely, just impeded in its growth.<br />
<br />
<br />
====Interpretation====<br />
<br />
This result makes intuitive sense, because as the function of the cell is repressed the cell produces less of the inhibiting siRNA, which leads to a regeneration of the cell. Eventually a steady state at a lower vitality is reached, where the vitality stays constant.<br />
<br />
==== Stochastic Model ====<br />
<br />
[[File:TUM13_SiRNA_stoch.png|thumb|right|400px| '''Figure 2''': Averaging over 1000 realisations of the Gillespie algorithm. Solid line indicates the mean, dashed lines indicate the 1-&sigma; interval.]]<br />
<br />
As number of siRNA in a cell can be very small, we cannot always assume that there is an sufficient amount of RNA such we can rule out stochastic effect. To deal with this problem we set up an analogous stochastic model constisting of a Markov Jump Process of the two species with a total of 4 reactions.<br />
<br />
[[File:TUM13_SiRNA_stoch_system.png|center|400px]]<br />
<br />
<br />
The state space [0,1] was discretised to [1,2,3,...,100] for both species R and N while the reaction rates were kept the same.<br />
<br />
To simulate the model we used and implementation of the Gillespie Algorithm with 1000 realisations and then averaged over all realisations.<br />
<br />
With this implementation the qualitative behaviour of the system remains unchanged, while the quantitative changes which is due to the discretization.<br />
<br />
==Nuclease Model==<br />
<br />
====Governing equations====<br />
[[File:TUM13_nuc_graph.png|thumb|right|400px| '''Figure 3:''' Example solution to our nuclease model with parameters p<sub>1</sub>=1, p<sub>3</sub>=0.5, p<sub>4</sub>=0.005.]]<br />
We determined the governing equations of this model to be the following:[[File:TUM13_nuc_formula.png|center]]<br />
<br />
with initial conditions V(0) = 1 and N(0) = 0, where at the time t=0 the trigger is activated.<br />
<br />
Figure 2, created by our MatLab script [[#nuclease_model_script|nuclease model]] shown below, on the left shows a solution to this system for some example parameters. It clearly shows that in this case the vitality of the cell decreases to 0 and remains there, i.e. that the cell has died.<br />
<br />
In the following we will verify, that this is the case for all parameter values.<br />
<br />
====Calculation of stable points and analysis====<br />
<br />
Any steady state given by V* and N* of this system must to satisfy [[File:TUM13_nuc_stable_satisfy.png|center]]<br />
<br />
<br />
The Hessian matrix of this system for the steady point is easy to solve [[File:TUM13_nuc_hessian_ev.png|center]]<br />
<br />
<br />
The eigenvector corresponding to the zero eigenvalue is [[File:TUM13_nuc_eigenvec.png]]. It is obvious from the equations that any disturbance away from the steady state along this vector will decay back to the steady state, '''so this is a stable attractor'''.<br />
<br />
====Results of our model====<br />
<br />
The '''nuclease''' always reduces the vitality of the moss to 0, i.e. '''kills it off completely'''.<br />
<br />
<br />
====Interpretation====<br />
<br />
Again this result is very intuitive, since the '''moss cannot regenerate''' from the destruction of its genome.<br />
<br />
===Conclusions===<br />
<br />
For a functional kill-switch it is necessary, that the cells are actually killed and not just live on with reduced vitality. So based on our modeling results the siRNA approach is not satisfactory, while the '''nuclease satisfies the requirement'''. '''As a result the team pursued the nuclease approach leading to our final kill-switch.'''<br />
<br />
<br />
==MatLab Scripts==<br />
<br />
====siRNA model script====<br />
<br />
<nowiki>% f = @(R,V,k) [k(3)* V - k(4) * R , -k(1) * R * V + k(2) *(V -1)* (R -1)];<br />
g = @(k,y) [k(3)* y(2)- k(4)*y(1); -k(1)*y(1)*y(2)+k(2)*(y(2)-1)*(y(1)-1)];<br />
<br />
%k= [ k1, k2, k3, k4 ]; %insert the appropriate reaction rate constant<br />
k = [ 1, 2, 2, 1];<br />
<br />
[TOUT, YOUT] = ode45(@(t,y) g(k,y) ,[0,10],[0;1]);<br />
<br />
plot(TOUT, YOUT(:,1), 'markersize', 15, 'linewidth', 5)<br />
hold on; plot(TOUT, YOUT(:,2), 'r', 'markersize', 15, 'linewidth', 5); hold off<br />
<br />
legend('siRNA concentration','Vitality');<br />
xlabel('time');<br />
set(gca,'FontSize',24);<br />
set(gcf,'position', [100 100 600 600]);<br />
<br />
axis square<br />
set(gcf,'Color','w');<br />
export_fig siRNA_pic.png<br />
</nowiki><br />
<br />
==== Stochastic siRNA script ====<br />
<br />
<nowiki>%% 1 States<br />
% [R,V]<br />
x0 = [0,100];<br />
<br />
%% 2 Reactions<br />
% stoichiometric matrix<br />
S = zeros(4,2);<br />
<br />
%% 2.1 increase siRNA<br />
% R -> R + 1<br />
% rate k3 * V<br />
S(1,:) = [1,0];<br />
<br />
%% 2.2 degrade siRNA<br />
% R -> R - 1<br />
% rate k4 * R<br />
S(2,:) = [-1,0];<br />
<br />
%% 2.3 increase vitality<br />
% V -> V + 1<br />
% rate k2 * ( V - 1 ) * ( R - 1 )<br />
S(3,:) = [0,1];<br />
<br />
%% 2.4 decrease vitality<br />
% V -> V - 1<br />
% rate k1 * R * V<br />
S(4,:) = [0,-1];<br />
<br />
<br />
param = [ 1, 2, 1, 2];<br />
<br />
% rates<br />
acell = { @(t,x,k) k(3) * x(2)/100,<br />
@(t,x,k) k(4) * x(1)/100,<br />
@(t,x,k) k(2) * ( x(2) - 100 )/100 * ( x(1) - 100 )/100,<br />
@(t,x,k) k(1) * x(1) * x(2)/100};<br />
<br />
a = @(t,x,k) [feval(acell{1},t,x,k),feval(acell{2},t,x,k),feval(acell{3},t,x,k),feval(acell{4},t,x,k)];<br />
<br />
% time vector<br />
<br />
N_time = 100;<br />
<br />
tt = linspace(0,100,N_time);<br />
<br />
% number of runs<br />
N_repeat = 1000;<br />
<br />
% output<br />
X_runs = zeros(N_repeat,N_time,length(x0));<br />
<br />
<br />
%plotting<br />
figure(1)<br />
clf<br />
hold on<br />
<br />
for j = 1:N_repeat <br />
[X_runs(j,:,:)]=SSA(x0,S,a,tt,param);<br />
end<br />
<br />
figure<br />
hold on<br />
plot(tt,mean(X_runs(:,:,1)),'b')<br />
plot(tt,mean(X_runs(:,:,2)),'r')<br />
legend('siRNA','Vitality')<br />
for k = 1:length(x0)<br />
switch k<br />
case 1<br />
plot(tt,mean(X_runs(:,:,k)),'b')<br />
plot(tt,mean(X_runs(:,:,k))+sqrt(var(X_runs(:,:,k))),'--b')<br />
plot(tt,mean(X_runs(:,:,k))-sqrt(var(X_runs(:,:,k))),'--b')<br />
case 2<br />
plot(tt,mean(X_runs(:,:,k)),'r')<br />
plot(tt,mean(X_runs(:,:,k))+sqrt(var(X_runs(:,:,k))),'--r')<br />
plot(tt,mean(X_runs(:,:,k))-sqrt(var(X_runs(:,:,k))),'--r')<br />
end<br />
end<br />
<br />
find(X_runs(:,:,2)==0,1,'first')<br />
set(gcf,'Color','w')<br />
axis square<br />
export_fig siRNA_stochastic.png<br />
</nowiki><br />
<br />
====Gillespie====<br />
<br />
<nowiki>function [ xx, XX, TT ] = SSA( x0, S, a, tt , param)<br />
%SSA Summary of this function goes here<br />
% Detailed explanation goes here<br />
<br />
% initialise<br />
TT(1) = 0;<br />
XX(1,:) = x0;<br />
<br />
% step<br />
i = 1;<br />
while (TT(end)<tt(end));<br />
% increment step<br />
i = i + 1;<br />
<br />
%compute rates<br />
at = a(TT(i-1),XX(i-1,:),param);<br />
a0 = sum(at);<br />
<br />
for k = 1 : length(at)<br />
aj(k) = sum(at(1:k)/a0);<br />
end<br />
<br />
%sample time<br />
TT(i) = TT(i-1) + expinv(rand,1/a0);<br />
<br />
% select reaction that happens<br />
j = find(aj>rand,1,'first');<br />
<br />
% add reaction<br />
XX(i,:) = XX(i-1,:) + S(j,:);<br />
end<br />
<br />
xx = zeros(length(tt),length(x0));<br />
for j = 1:length(tt)<br />
xx(j,:) = XX(find(TT<=tt(j),1,'last'),:);<br />
end<br />
<br />
end<br />
</nowiki><br />
<br />
====Nuclease model script====<br />
<br />
<nowiki>% f = @(N,V,k) [p(3)* V - p(4) * N , -p(1) * N * V ];<br />
g = @(p,y) [p(3)* y(2)- p(4)*y(1); -p(1)*y(1)*y(2)];<br />
<br />
%p= [ p1, p2, p3, p4 ]; %insert the appropriate reaction rate constant<br />
p = [ 1, NaN, 1, 0.5 ];<br />
<br />
[TOUT, YOUT] = ode45(@(t,y) g(p,y) ,[0,20],[0;1]);<br />
<br />
plot(TOUT, YOUT(:,1), 'markersize', 15, 'linewidth', 5)<br />
hold on; plot(TOUT, YOUT(:,2), 'r', 'markersize', 15, 'linewidth', 5); hold off<br />
<br />
legend('Nuclease concentration','Vitality')<br />
xlabel('time');<br />
set(gca,'FontSize',24);<br />
set(gcf,'position', [100 100 600 600]);<br />
<br />
axis square<br />
set(gcf,'Color','w')<br />
export_fig nuc_pic.png</nowiki><br />
<br />
</div><br />
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</div><br />
</div><br />
<br />
{{Team:TU-Munich/TUM13_Footer}}</div>FlorianA