http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Jparrish2012.igem.org - User contributions [en]2024-03-28T18:49:08ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T03:29:14Z<p>Jparrish: </p>
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<span>home</span><br />
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<li><a href="https://2012.igem.org/Team:Groningen/market_research">market research</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Festivals">festivals</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/symposia">presentations</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/international_cooperation">collaboration</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Media_coverage">media coverage</a></li><br />
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</head><br />
<br />
<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/Media_coverageTeam:Groningen/Media coverage2012-10-27T03:22:40Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<html><br />
<head><br />
<style><br />
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</style><br />
<br />
<br><br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1 >iGEM Groningen 2012 in the media</z1><br><br />
</div><br />
</div><br />
</head><br />
<br />
<body><br />
<br />
<br><br><br />
<p class=margin align=center><br />
<i>Click on any of the text below to redirect to our appearance!</i><br><br />
</p><br />
<p class=margin><br />
<font size=4 color=#ff6700><b>Movies & radio</b></font><br />
</p><br />
<p class=list><br />
- <a href=http://www.unifocus.nl/site/pagina.php?id_item=104&tab=journaals target=_blank>Unifocus</a><br><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/__uCj4DMXNs" frameborder="0"></iframe><br><br><br />
- <a href=http://uk.webhosting.rug.nl/wordpress/2011/2012/10/17/rottend-vlees/ target=_blank>UK (Universiteits Krant)(Dutch)</a><br><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/I43HGi1p59s" frameborder="0"></iframe><br><br><br />
- <a href=http://stugtv.nl/blog/2012/10/10/stug-radio-9-oktober-2/ target=_blank>STUG Radio (Dutch), interview starts at timepoint 30:00</a><br><br />
<div style="margin-left: 190px"><object width="480" height="90"><param name="movie" value="//www.mixcloud.com/media/swf/player/mixcloudLoader.swf?feed=http%3A%2F%2Fwww.mixcloud.com%2Fstugtv%2Fstug-radio-9-oktober%2F&embed_uuid=a3af246f-6e26-4737-8655-a844fe2d52d8&stylecolor=953737&embed_type=widget_standard"></param><param name="allowFullScreen" value="true"></param><param name="wmode" value="opaque"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.mixcloud.com/media/swf/player/mixcloudLoader.swf?feed=http%3A%2F%2Fwww.mixcloud.com%2Fstugtv%2Fstug-radio-9-oktober%2F&embed_uuid=a3af246f-6e26-4737-8655-a844fe2d52d8&stylecolor=953737&embed_type=widget_standard" type="application/x-shockwave-flash" wmode="opaque" allowscriptaccess="always" allowfullscreen="true" width="480" height="90"></embed></object><div style="clear:both; height:3px;"></div><p style="display:block; font-size:12px; font-family:Helvetica, Arial, sans-serif; margin:0; padding: 3px 4px; color:#953737;"><a href="http://www.mixcloud.com/stugtv/stug-radio-9-oktober/?utm_source=widget&amp;utm_medium=web&amp;utm_campaign=base_links&amp;utm_term=resource_link" target="_blank" style="color:#953737; font-weight:bold;">STUG Radio 9 oktober</a><span> by </span><a href="http://www.mixcloud.com/stugtv/?utm_source=widget&amp;utm_medium=web&amp;utm_campaign=base_links&amp;utm_term=profile_link" target="_blank" style="color:#953737; font-weight:bold;">Stugtv</a><span> on </span><a href="http://www.mixcloud.com/?utm_source=widget&utm_medium=web&utm_campaign=base_links&utm_term=homepage_link" target="_blank" style="color:#953737; font-weight:bold;"> Mixcloud</a></p><div style="clear:both; height:3px;"></div></div><br />
</p><br />
<br><br />
<p class=margin><br />
<font size=4 color=#ff6700><b>Newspapers & magazines</b></font><br />
</p><br />
<p class=list><br />
- <a href=http://www.meppelercourant.nl/?n_id=262626 target=_blank>Meppeler Courant (Dutch)</a><br><br />
- <a href=http://uk.webhosting.rug.nl/wordpress/2011/2012/10/08/rug-team-beste-van-europa/ target=_blank>UK (Universiteits Krant - Independent Weekly for the University of Groningen) (Dutch)</a><br><br />
- <a href=http://uk.webhosting.rug.nl/wordpress/2011/2012/10/04/wedstrijdje-bacterien-verbouwen/ target=_blank>article in UK</a><br><br />
<img src=https://static.igem.org/mediawiki/2012/5/57/Groningen2012_GG.png align=right width=250>- <a href=http://uk.webhosting.rug.nl/wordpress/2011/2012/10/12/igem-team-we-stonden-te-springen/ target=_blank> interview in UK (Universiteits Krant)</a><br><br />
- <a href=http://www.texelsecourant.nl/texelsecourant/nl/voorpagina/fotoartikel/3,0,85038?rpID=31 target=_blank>Texelse Courant</a><br><br />
- <a href=http://www.rug.nl/corporate/nieuws/archief/archief2012/nieuwsberichten/iGEM2012 target=_blank>University of Groningen website</a><br><br />
- <a href=http://www.dvhn.nl/nieuws/groningen/article9440101.ece/Bacteriebouwers-naar-Boston target=_blank>Dagblad van het Noorden</a><br><br />
- <a href=https://static.igem.org/mediawiki/2012/0/06/IGEM_Groningen_RR_20121006_Lifeline_draft.pdf target=_blank>Lifeline magazine</a><br><br />
- Groninger gezinsbode<br><br />
- <a href=https://2012.igem.org/File:Groningen_RR_20120627_Faculty_newsletter.jpg target=_blank>University of Groningen Faculty Newsletter</a><br><br />
</p><br />
<p class=margin><br />
...coming soon:<br />
</p><br />
<p class=list><br />
- <a href=http://idun.nl/vereniging/commissies/lifeline target=_blank>Lifeline magazine</a><br><br />
- <a href=http://www.c2w.nl/ target=_blank>www.c2w.nl</a><br><br />
- <a href=http://www.detexelsekunstenaars.nl/weblog/ target=_blank>De Texelse kunstenaars</a><br><br />
<br><br />
<p><br />
<p class=margin><br />
<font size=4 color=#ff6700><b>Websites</b></font><br />
</p><br />
<p class=list><br />
- <a href=http://www.rug.nl/scienceLinx/nieuws/20121010_iGEM?lang=en target=_blank>ScienceLinx</a><br><br />
- <a href=http://www.oogtv.nl/2012/10/rug-studenten-laten-bacterie-werken/ target=_blank>Omroep Organisatie Groningen</a><br><br />
- <a href=http://stugtv.nl/blog/2012/10/09/bacterie-nu-ook-nuttig/ target=_blank>STUG TV</a><br><br />
- <a href=http://biotecture.nl/blog.html target=_blank>Biotecture.nl</a><br><br />
</p><br />
<br><br><br><br><br><br><br><br><br />
</body><br />
</html><br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiativeTeam:Groningen/Stop the food waste initiative2012-10-27T03:20:05Z<p>Jparrish: </p>
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<z1>Stop the food waste initiative</z1><br />
</div><br />
</div><br />
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<div style="position:absolute; left: 125px; top: 220px; z-index:-1;"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Groningen2012_AD20120915_StopFoodWaste.png" width="130" height="130"><br />
</div><br />
<p class="marginleft"><br><br><br><br />
The amount of food that is thrown away each day globally is tremendous - hundreds of millions of tons, adding up to 1.3 billion tons of food thrown away yearly <a href=#ref1>[1]</a>. Most of the food is thrown away in the production process, even though a large part of it is good to eat; the bananas are too straight, the potatoes are not round enough. We, as consumers, can do little about this, except for electing politicians who would care for such issues. There is, however, a lot of food thrown away by us, at our homes.<br />
</p><br />
<p class="margin"><br />
Living in big cities, we lost the relationship with our food that humans used to have for generations. We often buy half-processed food products to prepare them fast and eat in a hurry. We know a food product has gone bad once there's mold on it, but many of us just use the best-before dates as indicators of food freshness and edibility. The best-before dates are there for informing us about the quality of our food as it seems, simple enough, right?<br><br />
<br><br />
<b>Wrong.</b><br><br />
<br><br />
We had expiration dates, and they can still be found on many food products. Expiration dates told us the minimal date in which our food, if stored properly, could have gone bad. But Best Before dates, now that is something different. Best before dates refer to food quality, not if it is still good to eat, or not. Now, many of the readers might think they want good quality food, so that's what the date is for. Unfortunately, that's wrong again. Food quality might refer to anything: the colour of your yoghurt, the size of your corn flakes. The yoghurt might turn a bit less pink over time, but it doesn't change, it's still perfectly fine to get eaten. Still, many consumers will throw away food getting close to the best before dates as "gone bad" - even a couple of days before the date itself!<br><br />
According to the UK Waste & Resources Action Programme (WRAP), 33 percent of all food produced is wasted along the chill chain or by the consumer. At the same time, a large number of people get sick every year due to spoiled food. Hilary Benn, former British Secretary of State for the Environment, Food and Rural Affairs, claimed in 2009 <a href="#ref2">[2]</a>:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Groningen2012_ADHilaryBennWebsite.png"><br><br />
<br><br />
The other issue is the "if stored properly" phrase. You will find information about storing your food on every package. The food is controlled very precisely on it's way from factory to the store, and in the store. Then we pick it up, buy it, take it home - and there the non-controllable conditions start. It can make a difference if your food travelled with you for only 15 minutes before you put it in your fridge, or freezer, or if it took two hours.<br><br />
<br><br />
Our <b>iGEM Groningen 2012</b> team wanted to come up with an alternative solution. We want to employ bacteria which would sense when your food product has gone bad - and communicate it with colour. Similar systems, based on pH or chemical reactions, have been proposed for milk and fish.<br><br />
So, we decided to try with meat.<br><br />
<br><br />
<b>Why meat?</b><br><br />
Meat is a major cause of food poisoning. It might not be easy to tell if your meat is still fresh. Also, meat is relatively expensive compared to other food products, so many consumers buy it in bulk, and refrigerate or freeze part of it for later. Experienced cooks often have stickers handy for writing the date when the meat first arrived and to know how long it stayed in the fridge (or freezer). But not many of us, consumers, do so. We rely on those due dates stamped on the packages.<br><br />
The result: much of the food gets thrown away - even if it is still perfectly edible. A sticker - not with the date, but showing the current state of food freshness, like the Food Warden - could be the solution to both unnecessary food waste, as food poisonings.<br><br />
<br><br />
Raising awareness about food waste is important - not only at home, but also in the industry. We only have one planet, and we need to start to be smarter about our food production and waste if we want the food industry to be sustainable with the growing human population. At iGEM Groningen, we would like you to try a simple experiment and write down every food product you throw away because it's expired, or if you're not sure if it's still good, and see how much the list grows over a month.<br><br />
<br><br />
There are a couple of things, though, that you can do to reduce household food waste. One of the ways to achieve this is to learn how to properly store your food. Did you know that if you keep lettuce in water, like flowers, it will last twice as long as than when kept in the fridge? Another way is to learn how to re-use the things you normally throw away. Apple peel can make very nice tea flavoring, and the leftover carrot pulp from your homemade carrot juice can be used to bake carrot cake. There are plenty of examples on the internet, if you want to look for them - we listed a couple in the links section below. <br><br />
<br><br />
Changing the world might seem hard, but everyone can change their own habits at their own homes; and change the world one fridge at a time.<br><br />
<br><br />
More about food waste:<br><br />
- <a href="http://www.tristramstuart.co.uk/" target=_blank>Tristram Stuart's page</a><br><br />
- <a href="http://www.scp-knowledge.eu/sites/default/files/knowledge/attachments/LNV%20-%20Factsheet%20drieluik%20A4%20Voedselverspilling%20Eng.pdf" target=_blank>Fact Sheet: Food Waste in the Netherlands</a><br><br />
- <a href="http://www.eea.europa.eu/nl/ema-signalen/signalen-2012/close-ups/voedselafval" target=_blank>Voedselafval — European Environment Agency (EEA)</a><br><br />
- <a href="http://www.tastethewaste.nl/" target=_blank>http://www.tastethewaste.nl/</a><br><br />
- <a href="http://en.wikipedia.org/wiki/Food_waste" target=_blank>Wikipedia on Food Waste</a><br><br />
- <a href="http://england.lovefoodhatewaste.com/content/about-food-waste" target=_blank>Love Food Hate Waste</a><br><br />
- <a href="http://www.stopfoodwaste.ie/" target=_blank>Website with tips how to reduce food waste at home</a><br><br />
- <a href="http://www.thekitchn.com/search?q=food+waste" target=_blank>Popular cooking blog on food waste</a><br><br />
</p><br />
<p class="ref"><br />
<b>References:</b><br><br />
<a name="ref1"></a>[1] <a href="http://www.fao.org/docrep/014/mb060e/mb060e00.pdf" target=_blank>Gustavson, Jenny; Cederberg, Christel; Sonesson, Ulf; van Otterdijk, Robert; Meybeck, Alexandre (2011). <i>Global Food Losses and Food Waste</i></a><br><br />
<a name="ref2"></a>[2] <a href="http://www.independent.co.uk/environment/green-living/kitchen-bin-war-tackling-the-food-waste-mountain-1698753.html">Rachel Shields (2009). <i>Kitchen bin war: tackling the food waste mountain</i></a><br />
</p><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiativeTeam:Groningen/Stop the food waste initiative2012-10-27T03:19:36Z<p>Jparrish: </p>
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</div><br />
</div><br />
</body><br />
<div style="position:absolute; left: 125px; top: 220px; z-index:-1;"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Groningen2012_AD20120915_StopFoodWaste.png" width="130" height="130"><br />
</div><br />
<p class="marginleft"><br><br><br><br />
The amount of food that is thrown away each day globally is tremendous - hundreds of millions of tons, adding up to 1.3 billion tons of food thrown away yearly <a href=#ref1>[1]</a>. Most of the food is thrown away in the production process, even though a large part of it is good to eat; the bananas are too straight, the potatoes are not round enough. We, as consumers, can do little about this, except for electing politicians who would care for such issues. There is, however, a lot of food thrown away by us, at our homes.<br />
</p><br />
<p class="margin"><br />
Living in big cities, we lost the relationship with our food that humans used to have for generations. We often buy half-processed food products to prepare them fast and eat in a hurry. We know a food product has gone bad once there's mold on it, but many of us just use the best-before dates as indicators of food freshness and edibility. The best-before dates are there for informing us about the quality of our food as it seems, simple enough, right?<br><br />
<br><br />
<b>Wrong.</b><br><br />
<br><br />
We had expiration dates, and they can still be found on many food products. Expiration dates told us the minimal date in which our food, if stored properly, could have gone bad. But Best Before dates, now that is something different. Best before dates refer to food quality, not if it is still good to eat, or not. Now, many of the readers might think they want good quality food, so that's what the date is for. Unfortunately, that's wrong again. Food quality might refer to anything: the colour of your yoghurt, the size of your corn flakes. The yoghurt might turn a bit less pink over time, but it doesn't change, it's still perfectly fine to get eaten. Still, many consumers will throw away food getting close to the best before dates as "gone bad" - even a couple of days before the date itself!<br><br />
According to the UK Waste & Resources Action Programme (WRAP), 33 percent of all food produced is wasted along the chill chain or by the consumer. At the same time, a large number of people get sick every year due to spoiled food. Hilary Benn, former British Secretary of State for the Environment, Food and Rural Affairs, claimed in 2009 <a href="#ref2">[2]</a>:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Groningen2012_ADHilaryBennWebsite.png"><br><br />
<br><br />
The other issue is the "if stored properly" phrase. You will find information about storing your food on every package. The food is controlled very precisely on it's way from factory to the store, and in the store. Then we pick it up, buy it, take it home - and there the non-controllable conditions start. It can make a difference if your food travelled with you for only 15 minutes before you put it in your fridge, or freezer, or if it took two hours.<br><br />
<br><br />
Our <b>iGEM Groningen 2012</b> team wanted to come up with an alternative solution. We want to employ bacteria which would sense when your food product has gone bad - and communicate it with colour. Similar systems, based on pH or chemical reactions, have been proposed for milk and fish.<br><br />
So, we decided to try with meat.<br><br />
<br><br />
<b>Why meat?</b><br><br />
Meat is a major cause of food poisoning. It might not be easy to tell if your meat is still fresh. Also, meat is relatively expensive compared to other food products, so many consumers buy it in bulk, and refrigerate or freeze part of it for later. Experienced cooks often have stickers handy for writing the date when the meat first arrived and to know how long it stayed in the fridge (or freezer). But not many of us, consumers, do so. We rely on those due dates stamped on the packages.<br><br />
The result: much of the food gets thrown away - even if it is still perfectly edible. A sticker - not with the date, but showing the current state of food freshness, like the Food Warden - could be the solution to both unnecessary food waste, as food poisonings.<br><br />
<br><br />
Raising awareness about food waste is important - not only at home, but also in the industry. We only have one planet, and we need to start to be smarter about our food production and waste if we want the food industry to be sustainable with the growing human population. At iGEM Groningen, we would like you to try a simple experiment and write down every food product you throw away because it's expired, or if you're not sure if it's still good, and see how much the list grows over a month.<br><br />
<br><br />
There are a couple of things, though, that you can use to reduce household food waste. One of the ways to achieve this is to learn how to properly store your food. Did you know that if you keep lettuce in water, like flowers, it will last twice as long as than when kept in the fridge? Another way is to learn how to re-use the things you normally throw away. Apple peel can make very nice tea flavoring, and the leftover carrot pulp from your homemade carrot juice can be used to bake carrot cake. There are plenty of examples on the internet, if you want to look for them - we listed a couple in the links section below. <br><br />
<br><br />
Changing the world might seem hard, but everyone can change their own habits at their own homes; and change the world one fridge at a time.<br><br />
<br><br />
More about food waste:<br><br />
- <a href="http://www.tristramstuart.co.uk/" target=_blank>Tristram Stuart's page</a><br><br />
- <a href="http://www.scp-knowledge.eu/sites/default/files/knowledge/attachments/LNV%20-%20Factsheet%20drieluik%20A4%20Voedselverspilling%20Eng.pdf" target=_blank>Fact Sheet: Food Waste in the Netherlands</a><br><br />
- <a href="http://www.eea.europa.eu/nl/ema-signalen/signalen-2012/close-ups/voedselafval" target=_blank>Voedselafval — European Environment Agency (EEA)</a><br><br />
- <a href="http://www.tastethewaste.nl/" target=_blank>http://www.tastethewaste.nl/</a><br><br />
- <a href="http://en.wikipedia.org/wiki/Food_waste" target=_blank>Wikipedia on Food Waste</a><br><br />
- <a href="http://england.lovefoodhatewaste.com/content/about-food-waste" target=_blank>Love Food Hate Waste</a><br><br />
- <a href="http://www.stopfoodwaste.ie/" target=_blank>Website with tips how to reduce food waste at home</a><br><br />
- <a href="http://www.thekitchn.com/search?q=food+waste" target=_blank>Popular cooking blog on food waste</a><br><br />
</p><br />
<p class="ref"><br />
<b>References:</b><br><br />
<a name="ref1"></a>[1] <a href="http://www.fao.org/docrep/014/mb060e/mb060e00.pdf" target=_blank>Gustavson, Jenny; Cederberg, Christel; Sonesson, Ulf; van Otterdijk, Robert; Meybeck, Alexandre (2011). <i>Global Food Losses and Food Waste</i></a><br><br />
<a name="ref2"></a>[2] <a href="http://www.independent.co.uk/environment/green-living/kitchen-bin-war-tackling-the-food-waste-mountain-1698753.html">Rachel Shields (2009). <i>Kitchen bin war: tackling the food waste mountain</i></a><br />
</p><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/File:ID_RR_cost_estimation_sticker.pdfFile:ID RR cost estimation sticker.pdf2012-10-27T03:11:05Z<p>Jparrish: uploaded a new version of &quot;File:ID RR cost estimation sticker.pdf&quot;</p>
<hr />
<div></div>Jparrishhttp://2012.igem.org/Team:Groningen/market_researchTeam:Groningen/market research2012-10-27T02:30:31Z<p>Jparrish: </p>
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<z1 >Market research</z1><br><br />
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<p class="margin"><br><br><br />
We have constructed a prototype of the The Food Warden sticker, containing our modified <i>Bacillus subtillis</i>. It could be developed into a potential product. Therefore, we decided to conduct a market research survey where we asked the respondents questions about their general food habits and if they would be interested in the Food Warden as a possible product on the market. We also asked if they would consider using a GMO-containing sticker next to food as a safe practice. We used the <a href="http://students.sgizmo.com/" target="_blank">surveygizmo</a> survey engine for this task.<br />
</p><br />
<p class="orange">The respondents</p><br />
<p class="margin"><br />
<img src="https://static.igem.org/mediawiki/2012/2/28/Groningen2012_ADChartAge.png" align=right style="padding-left: 10px">We received a total of 338 responses to the survey. Most of our respondents (58.3%) were 16-25 years of age, 27.8% of age between 26 and 40, and 13.3% of the respondents were older than 40. Only few respondents (0.6%) were younger than 16 years of age.<br><br />
This portrays our respondent group as a young crowd, in fact we recruited many of our respondents from student body of our university (from multiple faculties - these are not only biology students).<br> We only asked our respondents if they are biologists or biology students in the second run of the survey, but since we used the same channels to ask for the responses, we can estimate that the total fraction of biologists and biology students in our survey is proportional to the one obtained in the second run: about 16.4% of our respondents were biologists or biology students. The exact demo- and geographical spread of our respondents can be seen in the full report (available for dowload at the bottom of the page).<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6f/Groningen2012_ADChartWhere.png" align=left style="padding-right: 10px; padding-top:5px;"><br />
<br>The vast majority (76%) of the respondents answered that they go grocery shopping on their own, and they cook on their own. Also, most of them (91.1%) buy their meat at the supermarket. The biggest group of the respondents buys meat two to three times a week (36.4%), or almost everyday (27.5%). Overall, more respondents buy food products in smaller amounts, only what they need immediately, even if it costs more (53.6%) compared to buying bigger ("family") packages to save money (46.4%). </p><br><br />
<p class="orange">Best before dates and food waste<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen2012_ADChartFoodWaste.png" align=right style="padding-left: 10px"></p><br />
<p class="margin">The opinions of the respondents on "best before" dates are split: 60.4% does not think that such dates give an accurate measure of food edibility, while 39.6% believe they do. <br />
According to our survey results, many of the respondents throw away food - if they are not certain if it is good - relatively rarely (once a month - 46.8%, never or almost never - 18.9%). However, roughly a third of the respondents admit they do that often (30.5% - few times a month, 3.9% - more than once a week).<br><br />
<p class="orange">Food Warden as a potential product</p><br />
<p class="margin">86% of the respondents would like to use a product like the Food Warden sticker to indicate whether their meat is good to eat or not, out of which 70.1% would like to be sure if their meat is safe to eat, and 11.8% would be able to save money through buying bigger meat packages.<br><br />
<img src="https://static.igem.org/mediawiki/2012/4/42/Groningen2012_ADChartPrice.png" align=left style="padding-right: 10px; padding-top:5px;"><br>Interestingly, 68.7% of all respondents are willing to pay extra for their meat to include such a product, 5-15 eurocents or more.<br><br />
Moreover, 82.5% of the respondents would like to see a product like Food Warden on the market; 11.3% like the concept, but wouldn't use the product; only 6.2% think such product is not needed.<br><br />
<br><br />
<br><br />
<p class="orange">GMOs next to food<img src="https://static.igem.org/mediawiki/2012/c/ca/Groningen2012_ADChartSafe.png" align=right style="padding-left: 10px"></p><br />
<p class="margin">Since we were asked by many people about public perception of using GMOs next to food, we asked our respondents a very important question: Considering all of our safety measures, would you think the product containing GMOs would be safe to use next to your food? 54% answered yes, and 38.8% were not certain and would need to learn more about the safety measures. Only 5.1% of all respondents do not think it is safe.<br><br />
<p class="orange">Conclusions</p><br />
<p class="margin">We were positively surprised how enthusiastically many of our respondents reacted to our concept and the project. Among the feedback, we got some very thoughtful ideas about features that Food Warden would need to have as a product, but also many heart-warming messages (we keep the original spelling, mostly):</p><br />
<i><p class=quote>There is nothing more harmful about the bacteria you used than one that might get on my food from whatever surce including myself, for as far as I know you are not using a bacteria that is pathological in any sense, and the proteins produced by the bacteria will not be harmful either. There is no biohazard.</p><br />
<p class=quote>Save consume with FoodWarden! This is GOLD if you succeed and keep on trying to conquer the market. Just go for it!</p><br />
<p class=quote>I find it a very elegant system, because it prevends us from thoughing away good perfectly fine food.</p><br />
<p class=quote>It should be an European standard. You never know what producer, re-seller or retailer did with 'best before' dates. And most meat doesn't have it. On the other side 'best before' dates are not really a deadlines, so a lot of food is wasted, because it had reached its 'best before' date, but it is still eatable</p><br />
<p class=quote>I like the idea, and I think a lot of people will benefit from the product. Especially people who throw away food too soon with the only reason that it is at the expire date but still fine to eat</p><br />
<p class=quote>something like that should be standard </p><br />
<p class=quote>It is very useful. I would like to see products associated with this idea.</p><br />
<p class=quote>Very good initative-hope it will be a hype on the market !</p><br />
<p class=quote>It's good to know for sure if your meat or other products have turned bad. This way you can discard the best before date and just watch the indicator. It would be especially cool if there would be different levels for the state of the meat, eg. yellow for OK, orange almost rotten etc. Further I would very much like such a sticker for fish.</p><br />
<p class=quote>I think it's a really good idea. I don't like to throw away food, and I think this sticker will help me to throw away less food (meat).</p></i><br />
<p class="margin">Even though most the respondents of our survey say they do not throw away food often, they would still be interested in our product. It is also very surprising to see that almost none of the respondents reject the product immediately because of GMOs, even though they would have to put it next to their food, in their fridge. We think that the perception of GMOs in the society is changing and is growing in acceptance, as long as it is closely watched and controlled. People realise that insulin for diabetic patients is produced by GMOs, and that many crops and vegetables are genetically engineered. Even among the oldest respondents (age 40+), 38.6% would consider the product safe, and 52.3% chose the answer "maybe".<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a4/Groningen2012_ADChartDemand.png" align=left style="padding-right: 10px; padding-top:5px;">In general, our respondents were interested in the product as a food safety indicator, even if they do not throw away large amounts of food before due date, even if they do not suffer from food poisoning often. Interestingly, most people would like to pay a considerable amount of money extra to have a sticker like that included with their meat, and that underlines that there is a need for a product like Food Warden on the market.</p><br />
<p class="margin" align=right><a href="https://static.igem.org/mediawiki/2012/1/1c/Summary_Report_FULL_-_Oct_25_2012_-_Food_Warden_-_iGEM_Groningen_2012.pdf" target=_blank>Click to download the full survey report</a><br><br />
<a href=https://2012.igem.org/File:Summary_Report_-_Oct_25,_2012_-_40%2B_Food_Warden_-_iGEM_Groningen_2012.pdf target=_blank>Click to download survey report for ages 40+</a><br />
</p><br />
<p class="orange">Cost estimation of the sticker</p><br />
<p class="margin">As our survey shows, two-thirds of our respondents are willing to pay a couple of eurocents extra to have a Food Warden sticker included in the package. Therefore, we estimated the costs of producing the 0.5mL sticker using the materials we chose and under the assumption that we grow the bacteria on our own using a batch fermenter setup. The total costs of a single sticker, excluding production plant set up costs, add up to roughly <b>3.5 eurocents</b>.</p><br />
<p class="margin" align=right><a href="https://2012.igem.org/File:ID_RR_cost_estimation_sticker.pdf">Click to download the full cost estimation</a><br />
</p><br><br><br />
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{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T02:17:55Z<p>Jparrish: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
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{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T02:15:53Z<p>Jparrish: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T02:15:28Z<p>Jparrish: </p>
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</head><br />
<br />
<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/ProjectTeam:Groningen/Project2012-10-27T02:00:42Z<p>Jparrish: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T01:59:47Z<p>Jparrish: </p>
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Completed after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><br />
<i>Hover your mouse over the pictures to see more!</i><br />
</p><br />
<br><br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br><br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><br />
<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><br />
<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br />
<br><br />
These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br />
<br><br><br><br />
</p><br />
</td><br />
<td class="accCap"><br />
<p class="small"><br />
<font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br />
<br><br><br><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-27T01:37:38Z<p>Jparrish: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Abstract</z1><br />
</div><br />
</div><br />
<table class="centertable"><br />
<tr><br />
<td class="margincell" align="right"><br />
<div class="bigcog" id="bigcogtopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td colspan="4"><br />
<p class="nomargin"><br />
Every year, one third of global food production -1.3 billion tons of food- is thrown away, partially due to the “best before” dating system.<br />
<z7>iGEM Groningen 2012</z7> seeks to provide an alternative method of assessing edibility: the <z7>Food Warden</z7>. It uses an <z7>engineered strain</z7> <br />
of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger<br />
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of<br />
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will <br />
be visible to the naked eye. For safe usage of the system, spores of our engineered strain are placed into one half of a semi-permeable <br />
<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows<br />
<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.<br />
<br />
</p><br />
</td><br />
<td class="margincell" align="left"><br />
<div class="bigcog" id="bigcogtopright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
</tr><br />
<tr><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="bigcog" id="bigcogbottomleft"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div><br />
</td><br />
<td align="left" ><br />
<div class="cogoverlay" id="cogoverlaytopleft"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right" ><br />
<div class="cogoverlay" id="cogoverlaytopright"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px"><br />
</div><br />
</td><br />
<td align="right"><br />
<div class="bigcog" id="bigcogbottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px"><br />
</div> <br />
</td><br />
<td class="margincell"><br />
<div class="cogoverlay" id="cogoverlaybottomright"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px"><br />
</div> <br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<div class="cte3"><br />
<div class="ctd3"><br />
<z2 >Undertaken after European Regionals</z2><br><br><br />
</div><br />
</div><br />
<p class="marginChecklist"><br />
<br><br />
<br><br />
<a href="https://2012.igem.org/Team:Groningen/in_development" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Tested a construct with downregulated promoter <i>WapA</i></a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Construct" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Performed enhanced construct characterization</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/Sticker" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Characterized the influence of oxygen on germination and growth within the sticker</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/market_research" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Expanded the scope of the market survey</a><br><br />
<a href="https://2012.igem.org/Team:Groningen/international_cooperation" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png"> Collaborated with Cambridge 2012</a><br><br />
<br><br />
</p><br />
<br />
<a name="MainAcc"></a> <br />
<div class="cte2"><br />
<div class="ctd2"><br />
<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p align=center style="color: white; font-size: 10pt;"><i>Hover your mouse over the pictures to see more!</i></p><br><br />
<br />
<table class="accompli"><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
<br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/7/74/Groningen2012_ADVolatilesBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><br><b>1.</b></font> We designed and tested the "<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>": a capsule in which bacteria are kept inside and volatiles can go through. We used a model to get insight on how to tweak the growth of <i>Bacillus subtilis</i>.</p><br />
</td><br />
<br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><b>2.</b></font> We explored the definition of spoiled meat and ways to check meat spoilage. We identified <a class="inlink" href="https://2012.igem.org/Team:Groningen/volatiles">various compounds</a> present in spoiled meat.<br></p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 /><img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
<br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><b>3.</b></font> We identified spoiled meat <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">sensors</a> by transcriptome analysis.<br><br><br></p><br />
</td><br />
<br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><b>4.</b></font> <a class="inlink" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K818000" target="_blank">Backbone pSac-Cm</a>: easy cloning in<br><i>B. subtilis</i>, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, <i>E. coli</i> compatible, stabily inserted into the <i>B. subtilis</i> chromosome.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td class="accPic"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 /><img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
<br />
<td class="accPic"><br />
<ul class="hoverbox" style="margin-left: 30px"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 /><img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 /></a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><br><br><b>5.</b></font> We made <a class="inlink" href="https://2012.igem.org/Team:Groningen/pigmentproduction">AmilCP and AmilGFP</a> suitable for expression in <i>Bacillus subtilis</i>.<br> These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br><br><br></p><br />
</td><br />
<br />
<td class="accCap"><br />
<p class="small"><font color=#FF6700><b>6.</b></font> Most importantly: we developed a <a class="inlink" href="https://2012.igem.org/Team:Groningen/Construct">construct</a> which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.<br><br><br></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br><br><br><br />
</body><br />
<br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-27T01:18:07Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1 >Kill switch</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
<br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
short amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
<br><br />
<br><br />
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</body><br />
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<br />
{{Template:SponsorsGroningen2012}}<br />
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<div style="position:absolute; right: 0px; bottom: 760px;"><br />
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</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
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</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-27T00:58:10Z<p>Jparrish: </p>
<hr />
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<div class="cte"><br />
<div class="ctd"><br />
<z1 >Kill switch</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
<br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
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<html><br />
<a href="https://2012.igem.org/Team:Groningen/Modeling"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-27T00:55:53Z<p>Jparrish: </p>
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<div>{{HeaderGroningen2012}}<br />
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<div class="ctd"><br />
<z1 >Kill switch</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
<br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Modeling"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-27T00:44:21Z<p>Jparrish: </p>
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<div class="ctd"><br />
<z1 >Kill switch</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<br><br />
<p><br />
<br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
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</body><br />
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{{Template:SponsorsGroningen2012}}<br />
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<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-27T00:01:20Z<p>Jparrish: </p>
<hr />
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<z1 >Kill switch</z1><br />
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<br><br />
<p><br />
<br><br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
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{{Template:SponsorsGroningen2012}}<br />
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</html></div>Jparrishhttp://2012.igem.org/Template:HeaderGroningen2012Template:HeaderGroningen20122012-10-27T00:00:47Z<p>Jparrish: </p>
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<hr />
<div>#REDIRECT [[Team:Groningen/Kill Switch]]</div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-26T23:59:36Z<p>Jparrish: moved Team:Groninge/Kill Switch to Team:Groningen/Kill Switch: Typo</p>
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<z1 >Kill switch</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
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<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a<br />
pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by <br />
tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br />
<br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br />
<br><br />
The core concept behind the Food Warden is that it should pave the way to a more comprehensive, scientifically informed prediction <br />
of food edibility that goes beyond conventional best-before dates. The Food Warden as it is now is only a proof of principle. <br />
The goal is to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device <br />
requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br />
<br><br />
<br><br />
<ol class="indentlist"><br />
<li>Volatile concentration: Building upon our gas chromatography approach in order to quantitatively assess the volatile production of spoiling meat.</li><br />
<li>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food <br />
(see <a class="inlink" href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative">Stop the food waste initiative</a>) make this a difficult step. <br />
A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br />
</li><br />
<li>Pigment production: The control of pigment production dynamics will depend on the outcome of the previous two aspects of the <br />
tuning procedure. Once the relationship between volatile composition/concentration and health risk is elucidated to some degree, the pigment production can be <br />
tuned to fit this parameter. </li><br />
</ol><br />
</p><br />
<p><br />
<br><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a positive feedback system to<br />
increase the pigment production. One example of the positive feedback system that can be applied to increase pigment production under the regulation of the <br />
rotten meat promoter is shown below:<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Groningen2012_EJ_20120924_positive_feedback_construct.png" width=400 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Groningen2012_EJ_20120924_positive_feedback_construct.png" class="preview" width=700 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way that the pigment and the<br />
inducer will be in the loop, increasing the production rate of the pigment. This system is meant to increase the production speed of the pigment. <br />
One of the possible set of inducible promoter-inducer is pRE promoter (BBa_K116603) with CII (BBa_K116602). <br />
<br><br />
<br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br />
<br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system in terms of user-friendliness <br />
that need to be addressed. The Food Warden can only do its job if it can grow properly upon breaking of the inner compartment of the sticker.<br />
It is plausible that manufacturing errors during the production of an eventual Food Warden product could lead to issues with the germination of the spores, <br />
resulting in a sticker that does not do its job. Eventualities include:<br />
<br><br />
<br><br />
<ol class="indentlist"><br />
<li>Incorrect medium: In the event that the medium supplied in the sticker is not of the correct composition, <br />
the Food Warden will not grow, and therefore will not be able to identify spoiling meat. </li><br />
<li>Absence of viable spores: A defect sticker could be accidentally produced that either lacks spores capable <br />
of germination or lacks spores entirely.</li><br />
<li>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated <br />
with another organism.<br />
If the user is not aware of these problems he or she may assume that the lack of pigment production simply means the meat in question is not spoiling yet, <br />
whereas it may already be spoiling and indeed will without any warning.<br />
</ol><br />
</p><br />
<p><br />
<br><br />
<br><br />
Although these eventualities are a production process concern, it is our job to produce a system that minimizes the risk of these problems affecting the user. <br />
Therefore, we would have liked to build in a positive control that allows the user to confirm that the Food Warden in their sticker germinates and grows. <br />
The positive control considered was the inclusion of a constitutively produced second pigment. This pigment serves as a signal to the user that the Food Warden <br />
is functional upon germination. This positive control pigment should of course not interfere with the visual detection of the warning pigment. To ensure this,<br />
the following options were considered:<br />
<br><br />
<br><br />
<ol class="indentlist"><br />
<li>Choosing the two pigment colors such that the warning pigment color is highly dominant over the control pigment color.</li><br />
<li>Placing the pigment under the control of a weak constitutive promoter, producing the control pigment at minimum levels <br />
required for user detection, therefore more easily being overpowered by the warning pigment.</li><br />
<li>Placing the pigment under the control of a constitutive promoter, identified in our microarray analysis, that downregulates<br />
the gene it controls under spoiling meat conditions, allowing the warning pigment to more easily overpower the control pigment <br />
(go to the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor">Sensor page</a> for more information on the downregulated genes and operons).</li><br />
<li><br />
Including an operator for the transcription of the control pigment that allows repression by a repressor protein. <br />
This repressor protein would be under the control of the same promoter responsible for warning pigment transcription. The construct can be put as following:<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><br />
<img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
</li><br />
<li><br />
Placing a control pigment that reacts to a specific compound to change its color. The control pigment is <br />
under the regulation of a constitutive promoter while the rotten meat promoter regulates the compound needed to change the control pigment's color.<br />
The construct can be put as following: <br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><br />
<img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
When the rotten meat promoter is activated, the compound needed to change the control pigment's color and the repressor will be produced. <br />
The control pigment production will be stopped and the existing control pigment will start to react to the compound, changing its color <br />
and becoming the warning pigment. This can be achieved using existing biobrick: BBa_K274100 (lycopene, red pigment) and adding the compound <br />
(CrtY) encoded by BBa_K118008 to make the red color from lycopene into yellow color (beta-carotene). Another construct using positive feedback<br />
loop for the warning-pigment-compound without stopping the control pigment production is as following:<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><br />
<img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
In this system, the control pigment will still be produced even when the rotten meat promoter activates. The production speed of the second pigment<br />
compound which reacts to the control pigment to create new color is enhanced by the positive feedback loop, so the warning pigment color can be <br />
immediately produced.<br />
<br><br />
<br><br />
</li><br />
</ol><br />
</p><br />
<p><br />
This idea basically mimics the traffic light function: different colors production for every state of the meat. When the meat is still fresh, a <br />
specific pigment will be produced. When the meat starts to rot, another pigment will be produced overriding the previous pigment. <br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we succeeded to make a new construct: AmilGFP under regulation of P<i>wap</i>A (rotten meat downregulated promoter). <br />
The construct is a pilot construct for the following diagram: pigment 1 is regulated by the rotten meat down-regulated promoter (wapA) and pigment<br />
2 is regulated by the up-regulated promoter (sboA).<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=400 /><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=700 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
When meat is still fresh, the P<i>wap</i>A will regulate a production of pigment 1 while pigment 2 (that is regulated by P<i>sbo</i>A) is absent. <br />
When the meat is rotten, P<i>wap</i>A will be down-regulated, thus decreasing the production of pigment 1 and P<i>sbo</i>A will be upregulated, <br />
producing pigment 2. For example: If pigment 1 is a yellow pigment (amilGFP) and pigment 2 is a blue pigment (amilCP), a strong yellow color will <br />
be produced when the meat is still fresh and when the meat is rotten, more blue pigment will be produced. So when the meat is rotten, green color<br />
pigment is obtained. <br />
<br><br />
<br><br />
<z2>Towards a psychotrophic chassis</z2><br />
<br><br />
<br><br />
A different bacterial chassis is needed for further application of our volatile detection system. This is due to the inability of <br />
<i>Bacillus subtilis</i> to grow in a low temperature environment. The main criteria our chassis must:<br />
<br><br />
<br><br />
- Grow in a low temperature environment (psychotropic bacteria).<br><br />
- Form endospores, essential for effective storage and our 'sticker' activation mechanism.<br><br />
- Exhibit non-pathogenic activity.<br />
<br><br />
<br><br />
Literature study suggests the use of psychrotrophic Gram-positive bacteria from the <i>Bacillus</i> family as the new bacterial chassis for the volatile <br />
detection system, especially due to their ability to form spores. Although new strains of psychrotrophic <i>Bacillus</i>, such as the soil dwelling<br />
<i>Bacillus hunanensis</i>, have been discovered recently, their possible pathogenic characteristics are not yet described in literature (Chen et al. 2011).<br />
Some <i>B. cereus</i> strains are known to be able to grow in <7°C (Dufrenne et al. 1995), while some strains (e.g. <i>B. cereus strain Toyoi</i>)<br />
have been widely used as probiotic (Taras et al. 2005) & (Duc et al. 2004). However, no strain of <i>B. cereus</i> was found to fit all the chassis criteria, as the<br />
psychrotrophic strains tend to be pathogenic. <br />
<br><br />
<br><br />
On the other hand, a study (Shehata, Collins 1971) reported a <i>B. subtilis strain</i>, RH22, exhibited a cold resistance phenotype when they were<br />
grown in 5°C. To further investigate and hopefully find our own psychrotrophic <i>Bacillus subzerus</i> strain (a *subtil..is* joke), we can combine <br />
the sequencing protocols as described in this study, with, in the case of <i>Bacillus cereus</i> relatives, the use of 16S rRNA sequence analysis<br />
for detailed identification of microorganisms (Bavykin et al. 2006).<br />
<br><br />
<br><br />
There have been studies where modulation of bacterial temperature tolerance has been achieved by introducing chaperonin genes from arctic bacterium <br />
Oleispira antarctica into <i>Escherichia coli</i>(Ferrer et al. 2003). Unfortunately, no published results were found that applied analogous strategies <br />
to <i>Bacillus</i> strains.<br />
</p><br />
<br><br />
<z4>References</z4> <br />
<br><br />
<p class="ref"><br />
<ol class="reflist"><br />
<li>Bavykin, S.G., Lysov, Y.P., Zakhariev, V., Kelly, J.J., Jackman, J., Stahl, D.A. & Cherni, A. 2006, "Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms (vol 42, pg 3711, 2004)", Journal of clinical microbiology, vol. 44, no. 7, pp. 2676-2676.</li><br />
<li>Chen, Y., Hao, D., Chen, Q., Zhang, Y., Liu, J., He, J., Tang, S. & Li, W. 2011, "Bacillus hunanensis sp nov., a slightly halophilic bacterium isolated from non-saline forest soil", Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, vol. 99, no. 3, pp. 481-488. </li><br />
<li>Duc, L., Hong, H., Barbosa, T., Henriques, A. & Cutting, S. 2004, "Characterization of Bacillus probiotics available for human use", Applied and Environmental Microbiology, vol. 70, no. 4, pp. 2161-2171. </li><br />
<li>Dufrenne, J., Bijwaard, M., Tegiffel, M., Beumer, R. & Notermans, S. 1995, "Characteristics of some Psychrotrophic Bacillus-Cereus Isolates", International journal of food microbiology, vol. 27, no. 2-3, pp. 175-183. </li><br />
<li>Ferrer, M., Chernikova, T., Yakimov, M., Golyshin, P. & Timmis, K. 2003, "Chaperonins govern growth of Escherichia coli at low temperatures", Nature biotechnology, vol. 21, no. 11, pp. 1266-1267. </li><br />
<li>Shehata, T. & Collins, E. 1971, "Isolation and Identification of Psychrophilic Species of Bacillus from Milk", Applied Microbiology, vol. 21, no. 3, pp. 466-&. </li><br />
<li>Taras, D., Vahjen, W., Macha, M. & Simon, O. 2005, "Response of performance characteristics and fecal consistency to long-lasting dietary supplementation with the probiotic strain Bacillus cereus var. toyoi to sows and piglets", Archives of Animal Nutrition, vol. 59, no. 6, pp. 405-417. </li><br />
</ol><br />
</p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/acknowledgementsTeam:Groningen/acknowledgements2012-10-26T21:43:42Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<br />
<br />
<br />
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<head><br />
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</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Acknowledgments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<table class="margintable"><br />
<tr><br />
<td><br />
<table class="info"><br />
<tr colspan="2"><br />
<td class="textcell"><br />
<z5>We want to thank everybody who helped us during the project, especially:</z5><br />
</td><br />
</tr><br />
<tr><br />
<td class="textcell"><br />
<z6>Our supervisors and advisors</z6><br />
<br><br />
<br><br />
<z6>Leen van Wijngaarden</z6>, <z6>Jan Kiel</z6>, and <z6>Christa Holtkamp</z6> for making it possible that we could paint Bio art<br />
<br><br />
<br><br />
<z6>Ger Telkamp</z6> for providing us with lab equipment<br />
<br><br />
<br><br />
<z6>Sjoerd Murris</z6>, Science Linx - Summerschool<br />
<br><br />
<br><br />
<z6>Monique Smith</z6> and <z6>Prof. A.J. Minnaard</z6> for their help with the Gas Chromatography-Mass Spectrometry experiments and the data analysis<br />
<br><br />
<br><br />
We were advised by <z6>Dr. J. Lolkema</z6> and <z6>Prof. dr. ir. J.D van Elsas</z6> concerning our safety page<br />
<br><br />
<br><br />
<z6>Gert-Jan Euverink</z6> for sharing ideas on the sticker material<br />
<br><br />
<br><br />
<z6>Jan-Willem Veening</z6>, <z6>Jeroen Siebring</z6>, <z6>Tonia</z6>, <z6>Katrin</z6> because of their advice how to use the fluorescence microscope<br />
<br><br />
<br><br />
<z6>Anne de Jong</z6>, who advised us on the microarray experiments<br />
<br><br />
<br><br />
<z6>Anne Hesseling</z6>, for her help with finances<br />
<br><br />
<br><br />
<z6>Martijn Herber</z6>, <z6>Wout Overkamp</z6>, and many others of Molecular Genetics<br />
<br><br />
<br><br />
<z6>The Molecular Microbiology Group of the RUG</z6>, for letting us borrow the peristaltic pump and providing us with fresh impact on our project presentation<br />
<br><br />
<z6>Marten Staal</z6> and <z6>Marja van Leeuwen</z6> (Plant Physiology) for their help on the oxygen test set up.<br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/NotebookTeam:Groningen/Notebook2012-10-26T21:35:58Z<p>Jparrish: </p>
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<div class="ctewet"><br />
<div class="ctdwet"><br />
<z1>Wetwork</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
</tr><br />
<tr><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5June2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6June2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7June2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8June2012">8</a></td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11June2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12June2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13June2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14June2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18June2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19June2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20June2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21June2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22June2012">22</a></td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25June2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26June2012">26</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_27June2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28June2012">28</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_29June2012">29</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30June2012">30</a></td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1July2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2July2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3July2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4July2012">4</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5July2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6July2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7July2012">7</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8July2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_9July2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10July2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11July2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12July2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13July2012">13</a></td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16July2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17July2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18July2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19July2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20July2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21July2012">21</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22July2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23July2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24July2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25July2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26July2012">26</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_27July2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28July2012">28</a></td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30July2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<td colspan=4><br />
<table style="margin: 0 auto; background-color: transparent;"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1August2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2August2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3August2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4August2012">4</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5August2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6August2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7August2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8August2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_9August2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10August2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11August2012">11</a></td><br />
</tr><br />
<tr><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13August2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14August2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15August2012">15</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16August2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17August2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18August2012">18</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19August2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20August2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21August2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22August2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23August2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24August2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25August2012">25</a></td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28August2012">28</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_29August2012">29</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30August2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_31August2012">31</a></td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1September2012">1</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2September2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3September2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4September2012">4</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5September2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6September2012">6</a></td><br />
<td class="day">7</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8September2012">8</a></td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10September2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11September2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12September2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13September2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14September2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15September2012">15</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16September2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17September2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18September2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19September2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20September2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21September2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22September2012">22</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23September2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24September2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25September2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26September2012">26</a></td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>October</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
</tr><br />
<tr><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10October2012">10</a></td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
</tr><br />
<tr><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15October2012">15</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16October2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17October2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18October2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19October2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20October2012">20</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21October2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22October2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23October2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24October2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25October2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26October2012">26</a></td><br />
<td class="day">27</td><br />
</tr><br />
<tr><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<p class="z8"><br />
The protocols we used daily, can be found <a class="inlink" href="https://2012.igem.org/Team:Groningen/protocol">here</a>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<div class="ctemol"><br />
<div class="ctdmol"><br />
<z1>Modeling</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>March</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
</tr><br />
<tr><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
</tr><br />
<tr><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_16March2012">16</a></td><br />
<td class="day">17</td><br />
</tr><br />
<tr><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
</tr><br />
<tr><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
</tr><br />
<tr><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_5June2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_6June2012">6</a></td><br />
<td class="day">7</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_8June2012">8</a></td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_11June2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_12June2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_19June2012">19</a></td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_9July2012">9</a></td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_17July2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_18July2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_24July2012">24</a></td><br />
<td class="day">25</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_26July2012">26</a></td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_3August2012">3</a></td><br />
<td class="day">4</td><br />
</tr><br />
<tr><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_10August2012">10</a></td><br />
<td class="day">11</td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_12August2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_15August2012">15</a></td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
</tr><br />
<tr><br />
<td class="day">19</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_20August2012">20</a></td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_30August2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_31August2012">31</a></td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
</tr><br />
<tr><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_3September2012">3</a></td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_5September2012">5</a></td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_14September2012">14</a></td><br />
<td class="day">15</td><br />
</tr><br />
<tr><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
</tr><br />
<tr><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>October</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
</tr><br />
<tr><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
</tr><br />
<tr><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
</tr><br />
<tr><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
</tr><br />
<tr><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br />
<div class="ctehum"><br />
<div class="ctdhum"><br />
<z1>Human Practice</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>March</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
</tr><br />
<tr><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
</tr><br />
<tr><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16March2012">16</a></td><br />
<td class="day">17</td><br />
</tr><br />
<tr><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
</tr><br />
<tr><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_12April2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16April2012">16</a></td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_27April2012">27</a></td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_3May2012">3</a></td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_21May2012">21</a></td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24May2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_25May2012">25</a></td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_31May2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_1June2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_2June2012">2</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_3June2012">3</a></td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_7June2012">7</a></td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_11June2012">11</a></td><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_13June2012">13</a></td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_21June2012">21</a></td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<td colspan=4><br />
<table style="margin: 0 auto; background-color: transparent;"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16July2012">16</a></td><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_18July2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_1August2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_2August2012">2</a></td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
</tr><br />
<tr><br />
<td class="day">5</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_6August2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_7August2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_8August2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_9August2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_10August2012">10</a></td><br />
<td class="day">11</td><br />
</tr><br />
<tr><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_13August2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_14August2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_15August2012">15</a></td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
</tr><br />
<tr><br />
<td class="day">19</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_20August2012">20</a></td><br />
<td class="day">21</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_22August2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_23August2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24August2012">24</a></td><br />
<td class="day">25</td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
</tr><br />
<tr><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_5September2012">5</a></td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_10September2012">10</a></td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
</tr><br />
<tr><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_18September2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
</tr><br />
<tr><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24September2012">24</a></td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_27September2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_28September2012">28</a></td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
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</table><br />
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<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/NotebookTeam:Groningen/Notebook2012-10-26T21:35:31Z<p>Jparrish: </p>
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.calendarheader{<br />
background-color: rgba(0,0,0,0.3);<br />
text-align: center;<br />
color: white;<br />
font-weight: 500;<br />
border-width: 0px; <br />
width: 176px;<br />
margin-left: 2px;<br />
}<br />
.monthheader{<br />
background-color: transparent;<br />
border-style: none; <br />
}<br />
.dayheader{<br />
background-color: transparent;<br />
border-style: none;<br />
} <br />
.daylabel{<br />
font-size: 12pt;<br />
line-height: 12pt;<br />
background-color: rgba(0,0,0,0.3);<br />
width: 14%;<br />
border-color: transparent;<br />
border-style: none;<br />
border-width: 0px;<br />
margin: 2px;<br />
margin-color; transparent;<br />
}<br />
.daylabelend{<br />
font-size: 12pt;<br />
line-height: 12pt;<br />
background-color: rgba(0,0,0,0.3);<br />
width: 14%;<br />
border-color: transparent;<br />
border-style: none;<br />
border-width: 0px;<br />
}<br />
.day{<br />
font-size: 12pt;<br />
line-height: 12pt;<br />
background-color: rgba(0,0,0,0.3);<br />
width: 14%;<br />
}<br />
.daylink{<br />
background-color: rgba(0,0,0,0.6);<br />
width: 14%;<br />
}<br />
a.daylinka{<br />
font-size: 12pt;<br />
line-height: 12pt;<br />
color: rgb(255,103,0);<br />
}<br />
a.daylink:visited{<br />
font-size: 12pt;<br />
line-height: 12pt;<br />
color: rgb(255,103,0);<br />
}<br />
a.inlink{<br />
font-size: 10pt;<br />
line-height: 12pt;<br />
color: rgb(255,103,0);<br />
}<br />
table.centertable{<br />
background-color: transparent;<br />
width: 100%;<br />
margin-top: 0;<br />
margin-bottom: 0;<br />
}<br />
</style><br />
</head><br />
<br />
<body><br />
<div class="ctewet"><br />
<div class="ctdwet"><br />
<z1>Wetwork</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
</tr><br />
<tr><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5June2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6June2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7June2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8June2012">8</a></td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11June2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12June2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13June2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14June2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18June2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19June2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20June2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21June2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22June2012">22</a></td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25June2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26June2012">26</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_27June2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28June2012">28</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_29June2012">29</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30June2012">30</a></td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1July2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2July2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3July2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4July2012">4</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5July2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6July2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7July2012">7</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8July2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_9July2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10July2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11July2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12July2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13July2012">13</a></td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16July2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17July2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18July2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19July2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20July2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21July2012">21</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22July2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23July2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24July2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25July2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26July2012">26</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_27July2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28July2012">28</a></td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30July2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<td colspan=4><br />
<table style="margin: 0 auto; background-color: transparent;"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1August2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2August2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3August2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4August2012">4</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5August2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6August2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_7August2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8August2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_9August2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10August2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11August2012">11</a></td><br />
</tr><br />
<tr><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13August2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14August2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15August2012">15</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16August2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17August2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18August2012">18</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19August2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20August2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21August2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22August2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23August2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24August2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25August2012">25</a></td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_28August2012">28</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_29August2012">29</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_30August2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_31August2012">31</a></td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_1September2012">1</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_2September2012">2</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_3September2012">3</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_4September2012">4</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_5September2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_6September2012">6</a></td><br />
<td class="day">7</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_8September2012">8</a></td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10September2012">10</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_11September2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_12September2012">12</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_13September2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_14September2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15September2012">15</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16September2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17September2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18September2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19September2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20September2012">20</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21September2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22September2012">22</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23September2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24September2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25September2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26September2012">26</a></td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>October</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
</tr><br />
<tr><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_10October2012">10</a></td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
</tr><br />
<tr><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_15October2012">15</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_16October2012">16</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_17October2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_18October2012">18</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_19October2012">19</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_20October2012">20</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_21October2012">21</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_22October2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_23October2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_24October2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_25October2012">25</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Wetwork_26October2012">26</a></td><br />
<td class="day">27</td><br />
</tr><br />
<tr><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<p class="z8"><br />
The protocols we used daily, can be found <a class="inlink" href="https://2012.igem.org/Team:Groningen/protocol">here</a>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<div class="ctemol"><br />
<div class="ctdmol"><br />
<z1>Modeling</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>March</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
</tr><br />
<tr><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
</tr><br />
<tr><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_16March2012">16</a></td><br />
<td class="day">17</td><br />
</tr><br />
<tr><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
</tr><br />
<tr><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
</tr><br />
<tr><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_5June2012">5</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_6June2012">6</a></td><br />
<td class="day">7</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_8June2012">8</a></td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_11June2012">11</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_12June2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_19June2012">19</a></td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_9July2012">9</a></td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_17July2012">17</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_18July2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_24July2012">24</a></td><br />
<td class="day">25</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_26July2012">26</a></td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_3August2012">3</a></td><br />
<td class="day">4</td><br />
</tr><br />
<tr><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_10August2012">10</a></td><br />
<td class="day">11</td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_12August2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_15August2012">15</a></td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
</tr><br />
<tr><br />
<td class="day">19</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_20August2012">20</a></td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_30August2012">30</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_31August2012">31</a></td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
</tr><br />
<tr><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_3September2012">3</a></td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_5September2012">5</a></td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/Modeling_14September2012">14</a></td><br />
<td class="day">15</td><br />
</tr><br />
<tr><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
</tr><br />
<tr><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>October</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
</tr><br />
<tr><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
</tr><br />
<tr><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
</tr><br />
<tr><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
</tr><br />
<tr><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br />
<div class="ctehum"><br />
<div class="ctdhum"><br />
<z1>Human Practice</z1><br><br />
</div><br />
</div><br />
<table class="calendargroup"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>March</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
</tr><br />
<tr><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
</tr><br />
<tr><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16March2012">16</a></td><br />
<td class="day">17</td><br />
</tr><br />
<tr><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<br />
<td class="day">21</td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
</tr><br />
<tr><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>April</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_12April2012">12</a></td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16April2012">16</a></td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_27April2012">27</a></td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>May</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_3May2012">3</a></td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
</tr><br />
<tr><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
</tr><br />
<tr><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
</tr><br />
<tr><br />
<td class="day">20</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_21May2012">21</a></td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24May2012">24</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_25May2012">25</a></td><br />
<td class="day">26</td><br />
</tr><br />
<tr><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_31May2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>June</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_1June2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_2June2012">2</a></td><br />
</tr><br />
<tr><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_3June2012">3</a></td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_7June2012">7</a></td><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
</tr><br />
<tr><br />
<td class="day">10</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_11June2012">11</a></td><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_13June2012">13</a></td><br />
<td class="day">14</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_15June2012">15</a></td><br />
<td class="day">16</td><br />
</tr><br />
<tr><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_21June2012">21</a></td><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
</tr><br />
<tr><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
</tr><br />
</table><br />
</td><br />
<tr><br />
</tr><br />
<td colspan=4><br />
<table style="margin: 0 auto; background-color: transparent;"><br />
<tr><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>July</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day">1</td><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="day">5</td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
</tr><br />
<tr><br />
<td class="day">8</td><br />
<td class="day">9</td><br />
<td class="day">10</td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
</tr><br />
<tr><br />
<td class="day">15</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_16July2012">16</a></td><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_18July2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
</tr><br />
<tr><br />
<td class="day">22</td><br />
<td class="day">23</td><br />
<td class="day">24</td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
</tr><br />
<tr><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_31July2012">31</a></td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
<tr class="monthheader"><br />
<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>August</mh><br><br />
</div><br />
</div><br />
</td><br />
</tr><br />
<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
<tr><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_1August2012">1</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_2August2012">2</a></td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
</tr><br />
<tr><br />
<td class="day">5</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_6August2012">6</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_7August2012">7</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_8August2012">8</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_9August2012">9</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_10August2012">10</a></td><br />
<td class="day">11</td><br />
</tr><br />
<tr><br />
<td class="day">12</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_13August2012">13</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_14August2012">14</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_15August2012">15</a></td><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="day">18</td><br />
</tr><br />
<tr><br />
<td class="day">19</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_20August2012">20</a></td><br />
<td class="day">21</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_22August2012">22</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_23August2012">23</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24August2012">24</a></td><br />
<td class="day">25</td><br />
</tr><br />
<tr><br />
<td class="day">26</td><br />
<td class="day">27</td><br />
<td class="day">28</td><br />
<td class="day">29</td><br />
<td class="day">30</td><br />
<td class="day">31</td><br />
<td class="day"> </td><br />
</tr><br />
</table><br />
</td><br />
<td><br />
<table class="calendarheader"><br />
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<td colspan=7 ><br />
<div class="mte"><br />
<div class="mtd"><br />
<mh>September</mh><br><br />
</div><br />
</div><br />
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<tr class="dayheader"><br />
<td class="daylabelend">Su</td><br />
<td class="daylabel">M</td><br />
<td class="daylabel">Tu</td><br />
<td class="daylabel">W</td><br />
<td class="daylabel">Th</td><br />
<td class="daylabel">F</td><br />
<td class="daylabelend">Sa</td><br />
</tr><br />
</table><br />
<table class="calendar"><br />
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<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day"> </td><br />
<td class="day">1</td><br />
</tr><br />
<tr><br />
<td class="day">2</td><br />
<td class="day">3</td><br />
<td class="day">4</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_5September2012">5</a></td><br />
<td class="day">6</td><br />
<td class="day">7</td><br />
<td class="day">8</td><br />
</tr><br />
<tr><br />
<td class="day">9</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_10September2012">10</a></td><br />
<td class="day">11</td><br />
<td class="day">12</td><br />
<td class="day">13</td><br />
<td class="day">14</td><br />
<td class="day">15</td><br />
</tr><br />
<tr><br />
<td class="day">16</td><br />
<td class="day">17</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_18September2012">18</a></td><br />
<td class="day">19</td><br />
<td class="day">20</td><br />
<td class="day">21</td><br />
<td class="day">22</td><br />
</tr><br />
<tr><br />
<td class="day">23</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_24September2012">24</a></td><br />
<td class="day">25</td><br />
<td class="day">26</td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_27September2012">27</a></td><br />
<td class="daylink"><a class="daylinka" href="https://2012.igem.org/Team:Groningen/Notebook/PublicRelations_28September2012">28</a></td><br />
<td class="day">29</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
</body><br />
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<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T21:22:35Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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</head><br />
<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of PwapA<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
The pigment production activity of P<i>wap</i>A-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter <br />
(P<i>sbo</i>A) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of P<i>wap</i>A in the presence of <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<br><br />
sboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate (faint color formation after 2 days). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
We grew <i>B. subtilis</i> containing sboA-AmilGFP device in the setup overnight (16 hours) at 37 degrees Celsius. In the picture below, you can see the result:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
while the same strain that was grown without meat only showed very faint yellow color. Both <i>B. subtilis </i> wildtype in this setup did not express <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(left) From left to (right) Wildtype grown without meat, <i>B.subtilis</i>(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, <i>B.subtilis</i>(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.<br><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><br />
To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared <br />
the growth of <i>B. subtilis</i> sboA-AmilGFP strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray<br />
setup for 12 hours and measured OD (600 nm), absorbance (395 nm) and assayed the color of the cells when pelleted. Below you can see the results: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of <i>Bacillus subtilis</i> sboA-AmilGFP strain grown for 12 hours while subjected to spoiled meat, fresh meat, or no meat. <br><br />
(right) Visibility of yellow color of pelleted cells by eye. Assay done with 5 previously made pellets of different color intensities as a reference to ensure objectivity. <br />
</p> <br />
<br> <br />
<p><br />
<z5>AmilGFP</z5> and <z5>AmilCP</z5> both are <z5>fluorescent proteins</z5>. We decided to quantify the amount of AmilGFP inside our <i>Bacillus subtilis</i> <br />
strain when subjected to spoiled meat and without meat. As a positive control, we paired the AmilGFP coding gene to the <z5>strong <i>Bacillus subtilis</i> <br />
promoter rrnB</z5>. We measured the fluorescence, the OD and color of the pellet of all four test subjects during growth for 12 hours. The picture above <br />
shows the difference in fluorescence after twelve hours. It is clear that in the presence of volatiles that produced by the spoiled meat, the sboA promoter<br />
was highly upregulated, thus more amilGFP was expressed.<br />
Previous tests showed that the intensity of AmilGFP expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain that was exposed to fresh meat was the same as <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<i>Bacillus subtilis</i>, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top (left) 1) positive control: strong promoter rrnB with AmilGFP. 2) SboA-AmilGFP exposed to spoiled meat. 3)Wild type 4)SboA-AmilGFP grown without meat.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
Color of pellets of sboA-GFP in a no-meat environment (above) and exposed to spoiled meat (below)after 6 hours(6H), 8 hours (8H), 10 hours (10H), and 12 hours (12H).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
AmilCP is expressed less strongly in <i>Bacillus subtilis</i> than AmilGFP. On plate, not induced by volatiles, a faint blue-greyish color is visible after <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
However, after placing <i>Bacillus subtilis</i> in our sticker and exposing the sticker to rotten meat volatiles, it turned into a clear purple color.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
</p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Kill_Switch"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/ModelingTeam:Groningen/Modeling2012-10-26T21:20:47Z<p>Jparrish: </p>
<hr />
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</head><br />
<br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Modeling</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
What is modeling? This year it can mean building a model to describe the growth curve, it can mean describing a potential signaling pathway,<br />
or, it can mean an attempt at building an organism-scale model of <i>B. subtilis</i>. Each of these definitions is realized through a separate<br />
initiative. If you came directly to this page from the Project menu, then you may be more interested in the techniques and algorithms used in the models.<br />
Skip ahead to the presentation below, and feel free to request a copy of the source code. If you navigated to this page from the Growth section of the <br />
Sticker page, then you're probably expecting some sort of computer simulation predicting real-world growth behavior. Head on down to the Quantification of<br />
Initial Medium Composition. However, if you're interested in learning how to balance complexity with usefulness, read it all. You will see that<br />
complexity is introduced only where necessary (FBA to fill in literature gaps and discover relationships, the organism-scale model to try and discover other<br />
behavior from the GC and gene-expression data). Unless you're designing a Rube Goldberg machine, it is always best to keep things as simple as possible.<br />
<br><br />
<br><br />
</p> <br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Initiatives</z1><br><br />
</div><br />
</div><br />
<p><br />
This year, the modeling portion of the project was tasked with providing tangible support for the rest of the team. <br />
Theory is all well and good, but a project with such a limited timeframe required focus. The result was three major initiatives:<br />
<br><br />
<br><br />
<ul class="olist"><br />
<font color=#FF6700><b>1.</b></font> Quantify the initial medium composition which would:<br />
</ul><br />
<ul class="indentlist"><br />
<font color=#FF6700><b>a.</b></font> Germinate the spores<br />
<br><br />
<font color=#FF6700><b>b.</b></font> Raise the population to reporter-required levels within the smallest possible spoilage time-frame<br />
<br><br />
<font color=#FF6700><b>c.</b></font> Maintain an active population at that level for a prolonged period of time, avoiding the onset of dormancy or sporulation<br />
</ul><br />
<br><br />
<ul class="olist"><br />
<font color=#FF6700><b>2.</b></font> The creation of a succinct explanation of TnrA activation, according to current literature.<br />
</ul><br />
<br><br />
<ul class="olist"><br />
<font color=#FF6700><b>3.</b></font> The creation of a dynamic model for <i>B. subtilis</i> suitable for flux balance analysis which responds to environmental cues.<br />
</ul><br />
<br><br />
<br><br />
</p> <br />
<p> <br />
<z2>Quantification of Initial Medium Composition</z2><br />
<br><br />
<br><br />
<z3>Purpose</z3> <br />
<br><br />
<br><br />
The <i>B. subtilis</i> within the sticker must go through distinct phases under defined time constraints. The only way to control the amount of biomass, and its behavior, is through the initial medium concentration. The containment unit does not contain any time-release capsules for providing a controlled level of nutrients.<br />
<br><br />
<br><br />
<z3>Methods</z3> <br />
<br><br />
<br><br />
Refresh the page if the presentation does not appear. Alternatively, click <br />
<a class="inlink" href="http://docs.google.com/a/igemgroningen.com/presentation/d/115_fqrnYapGmftVng6t2mMoml6cprEZ5GX3PutVhlMc/present#slide=id.p13">here</a> <br />
. In compliance with the wiki freeze, Google Drive places an unalterable timestamp on the document. <br />
Access granted upon request as this requires a specific email address.<br />
<br><br />
<br><br />
</p><br />
<div class="pres"><br />
<iframe src="https://docs.google.com/presentation/embed?id=115_fqrnYapGmftVng6t2mMoml6cprEZ5GX3PutVhlMc&start=false&loop=false&delayms=30000" frameborder="0" width="480" height="389" allowfullscreen="true" mozallowfullscreen="true" webkitallowfullscreen="true"></iframe><br />
<br><br />
Yes, you can make this presentation full screen. Click the button between the slide number and the cog.<br />
<br><br />
<br><br />
</div><br />
<p> <br />
<z3>Constrained FBA Model</z3> <br />
<br><br />
<br><br />
The constrained model used for flux balance/variability analysis was developed by adding thermodynamic data from the eQuilibrator <br />
database to iBsu1103 developed by Henry <i>et al.</i>. The article provided the model in SBML format. Subsystem information was<br />
contained in the article's supplementary information. libSBML was used to load the SBML model into MATLAB. Custom functions were<br />
used to (1) assign subsystems to reactions, (2) correlate thermodynamic data with metabolites, (3) to calculate the Gibbs <br />
energies of reaction, and (4) to convert the model to GDX; the format required for running FBA and FVA in GAMS.<br />
<br><br />
<br> <br />
The resulting model was composed thusly:<br />
<br><br />
<table class="modeltable"><br />
<tr><br />
<td class="modelpart">Compartments:</td><br />
<td class="modelinfo" colspan="2"><br />
Cytosol and Extracellular<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart">Metabolites:</td><br />
<td class="modelinfo" colspan="2"><br />
1138 (852 have listed Gibbs energy of formation)<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart">Reactions:</td><br />
<td class="modelinfo" colspan="2"><br />
1681 = 1157 intracompartmental (763 with Gibbs energy of reaction) + 244 exchange with medium + 280 transport between compartments<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart">Reaction Subsystems:</td><br />
<td><br />
<ol class="modelinfo"><br />
<li>Amino Acids and Derivatives <br />
<li>Biomass<br />
<li>Carbohydrates<br />
<li>Cell Wall and Capsule<br />
<li>Cofactors, Vitamins, Prosthetic Groups, Pigments<br />
<li>Exchange<br />
</ol><br />
</td><br />
<td><br />
<ol class="modelinfo"><br />
<li value="7">Fatty Acids and Lipids<br />
<li value="8">Macromolecular Synthesis<br />
<li value="9">Membrane Transport<br />
<li value="10">Metabolism of Aromatic Compounds<br />
<li value="11">Nucleosides and Nucleotides<br />
<li value="12">Sulfur Metabolism<br />
</ol><br />
</td><br />
</tr><br />
</table><br />
<br><br />
</p><br />
<p> <br />
<z3>Results</z3> <br />
<br><br />
<br><br />
A picture is worth a thousand words. Below you will see the spreadsheet which encapsulates all of the relationships and calculations into a user-friendly format.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/9/90/Groningen2012_JP_20120920_BiomassOverview_v3.png"></img><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<br><br />
<br><br />
But, what does it mean? Well, lets take a look at the Input section.<br />
<br><br />
<table class="modeltable"><br />
<tr><br />
<td class="modelpart2">Temperature:</td><br />
<td class="modelinfo"><br />
The general operating temperature of the device in degrees Celsius<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">OD at the Start of Log Growth:</td><br />
<td class="modelinfo"><br />
This value is modified till the Final OD value is close to the desired value (explained below)<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Initial Amount of Potassium:</td><br />
<td class="modelinfo"><br />
Milligrams of potassium at the start<br />
</td><br />
</tr><br />
</table><br />
</p><br />
<p> <br />
We have the intermediate values either used to calculate the rest, or to simply provide extra information:<br />
<table class="modeltable"><br />
<tr><br />
<td class="modelpart2">Number of Spores:</td><br />
<td class="modelinfo"><br />
This is a measure of the number of spores which should be contained in the sticker<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Generation Time:</td><br />
<td class="modelinfo"><br />
Doubling time in minutes<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Biomass Production:</td><br />
<td class="modelinfo"><br />
Rate of biomass production in millimolar / grams of dry weight biomass / hour<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Potassium Uptake:</td><br />
<td class="modelinfo"><br />
Rate of potassium uptake in millimolar / grams of dry weight biomass / hour<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Length of Exponential Growth:</td><br />
<td class="modelinfo"><br />
Number of hours for which the cells are happily growing exponentially<br />
</td><br />
</tr><br />
</table><br />
</p><br />
<p> <br />
And, we have the direct values of interest:<br />
<table class="modeltable"><br />
<tr><br />
<td class="modelpart2">Final OD:</td><br />
<td class="modelinfo"><br />
This is an experimentally maximized value at which (1) there is enough biomass for the reporter to be visible, <br />
and (2) the growth stops because of a lack of potassium not because of cell-crowding <br />
(we must ensure volatile transport is not hindered by crowding)<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Required Glucose:</td><br />
<td class="modelinfo"><br />
Total amount of glucose required to support growth to the final OD, in grams<br />
</td><br />
</tr><br />
<tr><br />
<td class="modelpart2">Minimum NH4:</td><br />
<td class="modelinfo"><br />
Minimum total amount of nitrogen required to support growth to the final OD, in grams<br />
</td><br />
</tr><br />
</table><br />
</p><br />
<p> <br />
So, what is missing? The endpoints of the active sensing region. When does it start? When does it end? <br />
As stated in the slides above, this region starts when there is enough biomass for the reporter to be visible. <br />
We know the reporter works, we have seen it. However, the relationship between reporter opacity and amount of biomass has not been quantified.<br />
<br><br />
<br><br />
Also, if you look to the top of this page, this initiative should also adjust the initial amounts to avoid dormancy or sporulation at the final OD. <br />
This is quite the sticky wicket. It requires extensive testing for our specific conditions. The best we can find from sporulation studies in literature is that<br />
you can expect <i>B. subtilis</i> to start producing spores 5 hours after the end of the exponential growth phase.<br />
</p><br />
<br><br />
<z4>References</z4> <br />
<br><br />
<p class="ref"><br />
<ol class="reflist"><br />
<li>A. Flamholz, E. Noor, A. Bar-Even, and R. Milo, “eQuilibrator--the biochemical thermodynamics calculator,” Nucleic Acids Research, vol. 40, no. D1, pp. D770–D775, Nov. 2011.</li><br />
<li>C. S. Henry, J. F. Zinner, M. P. Cohoon, and R. L. Stevens, “iBsu1103: a new genome-scale metabolic model of Bacillus subtilis based on SEED annotations,” Genome Biology, vol. 10, no. 6, p. R69, Jun. 2009.</li><br />
<li>B. J. Bornstein, S. M. Keating, A. Jouraku, and M. Hucka, “LibSBML: an API Library for SBML,” Bioinformatics, vol. 24, no. 6, pp. 880–881, Feb. 2008.</li><br />
<li>E. Fischer and U. Sauer, “Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism,” Nature Genetics, vol. 37, no. 6, pp. 636–640, May 2005.</li><br />
<li>J-W. Veening, W. k. Smits, L. w. Hamoen, and O. p. Kuipers, “Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media,” Journal of Applied Microbiology, vol. 101, no. 3, pp. 531–541, 2006.</li><br />
<li>M. Dauner, T. Storni, and U. Sauer, “Bacillus Subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture,” J. Bacteriol., vol. 183, no. 24, pp. 7308–7317, Dec. 2001.</li><br />
</ol><br />
</p> <br />
<p><br />
<br><br />
<br> <br />
<z2>TnrA Activation</z2><br />
<br><br />
<br><br />
<z3>Purpose</z3> <br />
<br><br />
<br><br />
Prior to the successful microarray experiment conducted by the wetwork team, the proposed volatile sensing mechanism <br />
tied reporter activation to the metabolism of ammonium/ammonia (NH4/NH3+). The nitrogen metabolism in <i>B. subtilis</i> <br />
is a convoluted mesh of reactions and (in)activation complexes mostly controlled by the TnrA transcription factor. <br />
In order to observe the effect of NH4 uptake on the TnrA, it was necessary to have a concise behavioral diagram. <br />
Unfortunately, no such diagram existed. The diagram for nitrogen metabolism on the KEGG database identified most<br />
of what was involved, but did so in an unclear manner.<br />
<br><br />
<br><br />
<z3>Results</z3> <br />
<br><br />
<br><br />
The figure below used the behavioral information from literature to highlight the active and inactive pathways during <br />
ammonium uptake. In terms of the project, the creation of this diagram brought to light a critical problem for using <br />
TnrA to sense extracellular NH4/NH3+; TnrA is only active when glutamine synthetase (GS) is actively converting NH4 to <br />
glutamine. This in turn is regulated by cell growth, not the amount of NH4/NH3+ present. However, in one article it was<br />
observed that GS is also active under conditions of nitrogen limitation. If the medium contained the precise amount of<br />
glutamine necessary to support the initial growth phase, then the glutamine would be depleted at the sensing phase.<br />
GS would activate to try and create more glutamine. GS would only deactivate when the amount of extracellular NH4/NH3+ <br />
reached a level to remove the lack of nitrogen as factor limiting further growth. <br />
<br><br />
<br><br />
The real result? If this sensing pathway was selected, then the wetwork team would have to find the necessary levels of <br />
glutamine to exploit the edge of nitrogen limitation. Possible, but not exactly the easiest mechanism to implement for the consumer market.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/3/39/Groningen2012_JP_20120615_NH4-TnrA_Relationship_v2.png"></img><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<z4>References</z4> <br />
<br><br />
<p class="ref"><br />
<ol class="reflist"><br />
<li>KEGG Database, "Nitrogen Metabolism - Bacillus subtilis", Kanehisa Laboratories, Last Modified: July 23, 2010. http://www.kegg.jp/kegg-bin/show_pathway?org_name=bsu&mapno=00910</li><br />
<li>SubtiWiki, http://subtiwiki.uni-goettingen.de/wiki/index.php</li><br />
<li>K. Gunka and F. M. Commichau, “Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation,” Molecular Microbiology, vol. 85, no. 2, pp. 213–224, 2012.</li><br />
<li>N. Doroshchuk, M. Gelfand, and D. Rodionov, “Regulation of nitrogen metabolism in gram-positive bacteria,” Molecular Biology, vol. 40, no. 5, pp. 829–836, 2006.</li><br />
<li>Study Guide, Chem153C, University of California, Los Angeles. <br />
<br><font size="0.6em">http://vohweb.chem.ucla.edu/voh/classes%5Cspring10%5C153CID28%5C11AminoAcidBiosynthesisSQA.pdf</font></li><br />
</ol><br />
</p><br />
<p><br />
<br><br />
<br> <br />
<z2>Dynamic Model</z2><br />
<br><br />
<br><br />
<z3>Purpose</z3> <br />
<br><br />
<br><br />
For our purposes, the dynamic model seeks to provide an explanation for the gene expression observed in the microarray experiment.<br />
However, for the scientific community in general, a dynamic model would be enable phenotype prediction over time under varying <br />
environmental conditions. Such a model would also be able to predict phenotypes for knock-out mutant strains. Probabilistic <br />
integrative modeling (PROM) uses gene expression data across a wide variety of environmental conditions to quantify the link <br />
between the transcriptional-regulatory network and the stoichiometric metabolite-reaction matrix. In other words, PROM modulates <br />
the reaction fluxes by looking at the likelihood a given reaction is active given the current state of gene expression. This method<br />
has been shown to accurately predict growth phenotypes in knock-out strains of <i>E. coli</i> and <i>M. tuberculosis</i>. Unfortunately,<br />
PROM does not consider the current environmental cues. It considers a multitude of prior environmental cues through the gene expression <br />
data. As such, it is really only useful for predicting fluxes in knock-out strains. Despite this limitation, it is still a great <br />
starting point for building a dynamic model as it considers both the transcriptional-regulatory network and a constraint-based <br />
stoichiometric model. It does not rely on the sparse kinetic parameters used in ODE models.<br />
<br><br />
<br><br />
Enter integrative dynamic flux balance analysis (idFBA); it combines the signaling, transcriptional-regulatory, and metabolic networks.<br />
<br><br />
<br><br />
<z3>Results</z3> <br />
<br><br />
<br><br />
The flowchart below shows the progress of the feasibility study. We were able to build a suitable metabolic model, and to find the information<br />
necesssary for implementing the transcriptional-regulatory network using PROM. We were even able to obtain some idFBA scripts from an author of<br />
the article who happened to be a supervisor to the University of Virginia iGEM team. Unfortunately, the singalling network was a mystery. Sure, <br />
Groningen studies this network in <i>B. subtilis</i> quite extensively, but there still are too many gaps to be able to predict the Wetwork team's<br />
microarray data from their gas chromatography data.<br />
<br><br />
<br><br />
This initiative did not progress past the feasibility study.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_JP_20120807_Workflow_idFBA.png"></img><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<z4>References</z4> <br />
<br><br />
<p class="ref"><br />
<ol class="reflist"><br />
<li>N. E. Lewis, H. Nagarajan, and B. O. Palsson, “Constraining the metabolic genotype–phenotype relationship using a phylogeny of in silico methods,” Nature Reviews Microbiology, vol. 10, no. 4, pp. 291–305, Apr. 2012.</li><br />
<li>S. Chandrasekaran and N. D. Price, “Probabilistic integrative modeling of genome-scale metabolic and regulatory networks in Escherichia coli and Mycobacterium tuberculosis,” PNAS, vol. 107, no. 41, pp. 17845–17850, Oct. 2010.</li><br />
<li>J. Min Lee, E. P. Gianchandani, J. A. Eddy, and J. A. Papin, “Dynamic Analysis of Integrated Signaling, Metabolic, and Regulatory Networks,” PLoS Comput Biol, vol. 4, no. 5, p. e1000086, May 2008.</li><br />
</ol><br />
</p><br />
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{{Template:SponsorsGroningen2012}}<br />
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<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/Kill_SwitchTeam:Groningen/Kill Switch2012-10-26T21:06:26Z<p>Jparrish: </p>
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<div>{{HeaderGroningen2012}}<br />
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<div class="cte"><br />
<div class="ctd"><br />
<z1 >The sticker</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
Ideally, after the use of the Food Warden system we would like the bacteria to kill themselves when they are not useful anymore. <br />
This to ensure the safety of the sticker. As many iGEM teams before us, we thought about an internal kill switch from the start of<br />
our iGEM project. That is why we already discussed this briefly in the <br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/environment" target="_blank">safety page</a>.<br />
<br><br />
<br> <br />
Our future plan is to place a <i>Bacillus subtilis</i> specific toxin gene behind the promoter of a stress factor that responds to <br />
nutrition limitation of the <i>Bacillus subtilis</i>. It is most likely that the <i>Bacillus subtilis</i> cells will be influenced <br />
by nutritional stress because our sticker is a closed system and only an x amount of nutrients is available. For this kill switch, <br />
timing is really important: when the Food Warden bacterium is activated, it will germinate and respond to volatiles of meat that starts<br />
to spoil, providing the user with information about the freshness of the meat. However, the growth of the bacteria will continue after<br />
the consumer has used our product. Limiting factors for growth will present themselves in time, one of these being limitation of nutrients. <br />
Modeling could predict when the most optimal timepoint is achieved. At this time point the stress factor will be triggered and instead<br />
of the normal response, a toxin will be produced which kills the cells.<br />
<br><br />
<br><br />
We could also decide to use the Violacein pigment (BBa_k274002), this is a pigment that is naturally toxic to <i>Bacillus subtilis</i>. <br />
After the pigment production, the user knows that the meat has started to spoil and subsequently the cells are automatically killed due <br />
to the pigment. However, if the pigment is not produced, for instance when there is fresh meat available, this might be a disadvantage <br />
because the cells will continue to live.<br />
<br><br />
<br><br />
Besides using an internal kill switch, we also thought about alternative solution to kill the bacteria, by using a chemical reaction. <br />
Our first idea was to incorporate a third compartment inside the sticker, containing a antimicrobial substance. After using the sticker, <br />
the user breaks this third compartment, thereby mixing the desinfectant with the cells, which results in the killing of the cells. <br />
In this case, we should clearly mark the compartments of the sticker to avoid that the user breaks the wrong compartment before use.<br />
<br><br />
<br><br />
Our most outstanding idea was to apply microwaves on the sticker after its use. The waves probably will kill the bacteria within a <br />
large amount of time. However we did not test this solution. We need to find the correct amount of time for killing all the bacteria. <br />
At the same time melting of the plastic should be prevented...<br />
<br><br />
<br><br />
</p> <br />
<z4>References</z4><br />
<p class="ref"><br />
1. Production of Antibacterial Violet Pigment by Psychrotropic Bacterium RT102 Strain Yoshitoshi Nakamura*, Chikako Asada, and Tatsuro<br />
Sawada BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Volume 8, Number 1 (2003), 37-40, DOI: 10.1007/BF02932896<br />
</p><br />
<br><br />
<br><br />
<br><br />
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</body><br />
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<br />
{{Template:SponsorsGroningen2012}}<br />
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</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T20:59:40Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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</head><br />
<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of PwapA<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
The pigment production activity of P<i>wap</i>A-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter <br />
(P<i>sbo</i>A) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of P<i>wap</i>A in the presence of <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<br><br />
sboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate (faint color formation after 2 days). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
We grew <i>B. subtilis</i> containing sboA-AmilGFP device in the setup overnight (16 hours) at 37 degrees Celsius. In the picture below, you can see the result:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
while the same strain that was grown without meat only showed very faint yellow color. Both <i>B. subtilis </i> wildtype in this setup did not express <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(left) From left to (right) Wildtype grown without meat, <i>B.subtilis</i>(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, <i>B.subtilis</i>(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.<br><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><br />
To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared <br />
the growth of <i>B. subtilis</i> sboA-AmilGFP strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray<br />
setup for 12 hours and measured OD (600 nm), absorbance (395 nm) and assayed the color of the cells when pelleted. Below you can see the results: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of <i>Bacillus subtilis</i> sboA-AmilGFP strain grown for 12 hours while subjected to spoiled meat, fresh meat, or no meat. <br><br />
(right) Visibility of yellow color of pelleted cells by eye. Assay done with 5 previously made pellets of different color intensities as a reference to ensure objectivity. <br />
</p> <br />
<br> <br />
<p><br />
<z5>AmilGFP</z5> and <z5>AmilCP</z5> both are <z5>fluorescent proteins</z5>. We decided to quantify the amount of AmilGFP inside our <i>Bacillus subtilis</i> <br />
strain when subjected to spoiled meat and without meat. As a positive control, we paired the AmilGFP coding gene to the <z5>strong <i>Bacillus subtilis</i> <br />
promoter rrnB</z5>. We measured the fluorescence, the OD and color of the pellet of all four test subjects during growth for 12 hours. The picture above <br />
shows the difference in fluorescence after twelve hours. It is clear that in the presence of volatiles that produced by the spoiled meat, the sboA promoter<br />
was highly upregulated, thus more amilGFP was expressed.<br />
Previous tests showed that the intensity of AmilGFP expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain that was exposed to fresh meat was the same as <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<i>Bacillus subtilis</i>, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top (left) 1) positive control: strong promoter rrnB with AmilGFP. 2) SboA-AmilGFP exposed to spoiled meat. 3)Wild type 4)SboA-AmilGFP grown without meat.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
Color of pellets of sboA-GFP in a no-meat environment (above) and exposed to spoiled meat (below)after 6 hours(6H), 8 hours (8H), 10 hours (10H), and 12 hours (12H).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
AmilCP is expressed less strongly in <i>Bacillus subtilis</i> than AmilGFP. On plate, not induced by volatiles, a faint blue-greyish color is visible after <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
However, after placing <i>Bacillus subtilis</i> in our sticker and exposing the sticker to rotten meat volatiles, it turned into a clear purple color.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
</p><br />
<br><br />
<br><br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<a href="https://2012.igem.org/Team:Groningen/Kill_Switch"><br />
<div style="position:absolute; (right) 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; (right) 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/sponsor_levelsTeam:Groningen/sponsor levels2012-10-26T20:17:57Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br />
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<html><br />
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<style><br />
z1 { <br />
font-size:18pt;<br />
line-height:21pt;<br />
color:white;<br />
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font-size:14pt;<br />
font-weight: bold;<br />
color: rgb(255,103,0);<br />
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font-size:14pt;<br />
font-weight: bold;<br />
color: rgb(255,103,0);<br />
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width: 220px;<br />
text-align: center;<br />
background: #000;<br />
background-color: rgba(0,0,0,0.3);<br />
padding: 5px ;<br />
margin: 30px auto;<br />
color: #FFF;<br />
}<br />
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width: 280px;<br />
text-align: center;<br />
background: #000;<br />
padding-right:10px;<br />
padding-left:10px;<br />
margin: 0 auto;<br />
background-color: rgba(0,0,0,0.3);<br />
color: #FFF;<br />
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color: white;<br />
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Sponsorship Levels</z1><br><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
If you wish to become a sponsor contact us at team@igemgroningen.com! There are various arrangements to choose from:<br />
<br><br />
<br><br />
<br><br />
<z2>Platinum level</z2> (investment of >3.000€)<br />
<br><br />
<br><br />
Sponsor will be shown our gratitude in every possible way of promotion. <br />
In addition to the Gold level acknowledgements, you will be presented as one of our main sponsors on every <br />
piece of media and will be granted a logo in the corner of every presentation slide. Further publicity will <br />
gladly be discussed upon request, for example during our societal activities (e.g. at science/art festivals in Groningen etc.).<br />
<br><br />
<br><br />
<z2>Gold level</z2> (investment of >1.000€)<br />
<br><br />
<br><br />
Sponsors will be able to reach high calibre professionals from around the world in a variety of high-profile<br />
demonstrations of gratitude, including large logo displays and verbal mentioning during presentations. In addition <br />
to the Silver level acknowledgments below (even larger logo), gold level sponsors will enjoy being verbally mentioned<br />
at the end of our presentation (and on the last presentation slide), will have a logo on our flyers, and a large logo<br />
at the top of our poster. We can distribute company flyers at the two Jamborees.<br />
<br><br />
<br><br />
<z2>Silver level</z2> (investment of >500€)<br />
<br><br />
<br><br />
Sponsors will have the benefits of logo repetition and public acknowledgement in several settings. <br />
In addition to the Bronze level acknowledgments below (only with a larger logo), silver level sponsors will <br />
be mentioned in the final presentation, and will show their logo on the bottom of our main posters. <br />
<br><br />
<br><br />
<z2>Bronze level</z2> <br />
<br><br />
<br><br />
Sponsors will be thanked throughout each step of our work: via the website and upon request with a logo printed on<br />
our shirts/dresses worn by all team members as soon as available and surely during our final presentations.<br />
<br><br />
<br><br />
<z5>Both monetary and material support are welcome!</z5><br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/StickerTeam:Groningen/Sticker2012-10-26T20:14:30Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<br />
<br />
<br />
<html><br />
<head><br />
<style><br />
z1 { <br />
font-size:18pt;<br />
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font-size:14pt;<br />
font-weight: bold;<br />
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font-size:14pt;<br />
font-weight: bold;<br />
color: white;<br />
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font-size:14pt;<br />
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<div class="ctd"><br />
<z1 >The sticker</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
Not everyone likes free living, engineered bacteria next to their food. But in order to make our Food Warden<br />
system work properly, our bacteria should be able to react to the volatiles coming off the spoiling meat while it<br />
is located in, for instance, a meat package. We had a lot of discussions about how to achieve a very safe and <br />
easy-in-use solution, and this is what we came up with:<br />
<br><br />
<br> <br />
<i>Bacillus subtilis</i> would be an ideal candidate as a chassis for our genetically engineered construct because it <br />
has the ability to form endospores, a kind of dormant state that they use for survival. They can survive high levels<br />
of heat (>100 °C), drying, radiation, and many damaging chemicals and simply be brought back 'to life' under the influence<br />
of sufficient nutrients and water. This restorative process is germination. For the information, <br />
take a look <a class="inlink" href="http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=69">here</a> and <br />
<a class="inlink" href="http://www.pearsonhighered.com/pearsonhigheredus/educator/product/products_detail.page?isbn=0132324601">here</a>. <br />
Because of this ability to go dormant, our bacterium can be stored and activated when it is needed!<br />
<br><br />
<br> <br />
However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a <br />
containment device to store and activate the spores at the right time. For clarity: our team keeps calling it 'the sticker' all the time.<br />
The sticker consists of two nested compartments. The inner compartment contains a calibrated amount of nutrients, <br />
while the outer semi-permeable capsule contains the spores of our engineered strain. Breaking the barrier between the two compartments <br />
allows germination and growth of <i>Bacillus subtilis</i> cells. <br />
<br><br><br />
Now the properties of the material we use for our sticker come into play. The polymer we used is TPX®, also<br />
called polymethylpentene. This polymer is available as thin, transparent sheets. The advantage is that it is <br />
relatively cheap, strong, and capable of letting through volatiles. The radius of the pores in TPX® is between <br />
1 nm and 10 nm, which is at least 50x larger than the average badmeat-volatile, but still small enough to keep liquid <br />
and bacteria or spores in (see the figure below). More detailed information about the sticker design and its experiments, are stated below. <br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Siebring J. 2012 (unpublished)<br />
</p><br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/RR_20120825_tpx.PNG" width="300"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Comparison of the size of the TPX® pores, volatiles and <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
<z2>Design requirements</z2><br />
<br><br />
<br><br />
<z3>Material</z3><br />
<br><br />
<br><br />
The material should not break easily and should resist a human grip strength with a minimum of 40 pounds pinch and pressure. <br><br />
The product should be made of a material that is light and easy to handle. <br><br />
The material should be durable and inexpensive.<br> <br />
The volatiles and oxygen should penetrate through the material, while the spores, bacteria and liquid should stay inside.<br><br />
The material should fit in a meat package.<br><br />
The material should not be toxic or become toxic for the bacteria <br><br />
The material should be able to cope with a temperature of at least 125 degrees Celsius.<br />
<br><br />
<br><br />
<z3>Measurements</z3><br />
<br><br />
<br><br />
We want a visible feedback system for the human eye that should easy to understand for the consumer. <br><br />
The visible feedback should not degrade over time.<br />
<br><br />
<br><br />
<z3>Appearance</z3><br />
<br><br />
<br><br />
Product should be have attractive color(s) and recognizable shape.<br />
<br> <br />
<br><br />
<z3>Safety</z3><br />
<br><br />
<br><br />
The bacteria should not escape from the sticker nor harm the environment or the costumer. <br />
Therefore the product should provide adequate support and be reliable.<br />
<br><br />
<br><br />
<z3>Customer comfort</z3><br />
<br><br />
<br><br />
Easy to use the visible feedback device. <br />
<br><br />
The product may provide different degrees of strength depending on the development of the consumer strength. <br />
<br><br />
<br><br />
<z2>Development design</z2><br />
<br><br />
<br><br />
For the development of the sticker, the PDCA (Plan Do Check Act) strategy was used to develop an ultimate <br />
device that keeps track of the needs of the customer and the environment in the project design.<br />
<br><br />
<br> <br />
The following questions where raised: <br />
<br><br />
<br><br />
Who is your external customer?<br />
<br><br />
The customer who is against food spoilage and customers that are willing to buy it.<br />
<br><br />
<br> <br />
What value do you want to deliver to that customer?<br />
<br><br />
A replacement for the "use by date and sell by date" system. We want to develop a new product that <br />
indicates when meat starts to spoil before the costumer can notice this by eye, by smell or by taste.<br />
<br> <br />
<br><br />
Who, in you iGEM group, delivers that value?<br />
<br><br />
The design engineers and the biologists of iGEM Groningen 2012.<br />
<br><br />
<br><br />
How do they deliver that value?<br><br />
By doing research and creating a new indicator to predict when meat starts to spoil.<br />
<br><br />
<br><br />
<z3>Plan</z3><br />
<br><br />
<br><br />
Our first plan is to identify exactly what we have to do to make a sticker. <br />
The sticker should be the same as the use of a glow-in-the-dark stick, that you first have to<br />
break before it gives off a bright chemical light. Everyone should understand how <br />
it works. The indication color might be made with the same colors as a traffic light, to <br />
indicate when the meat is fresh, a bit spoiled or really spoiled. During our second case, <br />
we want to produce an easily activated sticker. In the third part of the plan, the outer sticker should be strong enough, <br />
let volatiles go through the outer layer of the sticker, and not any bacteria or liquid should go<br />
out of the outer layer of the sticker.<br />
<br><br />
<br><br />
</p><br />
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<br><br />
<br><br />
<br> <br />
<p><br />
<z3>Do</z3><br />
<br><br />
<br><br />
The following solution was chosen:<br />
Build a transparent plastic bag with inside a smaller compartment, where the medium inside is visible for the user when the color changes.<br />
Make the inner compartment of a weaker material where the sides break when a pinch force is applied.<br />
<br><br />
<br><br />
The following pilots where made:<br><br />
<br><br />
1. Make sticker user-friendly concerning the breakage of the inner compartment.<br><br />
2. Test if the bacteria grow in the sticker.<Br><br />
3. Test if the medium with the Food Warden bacteria will product a pigment in the sticker. <br />
<br><br />
<br><br />
<z3>Check</z3><br />
<br><br />
<br><br />
Depending on the success of the pilots, the number of areas for improvement you have identified, and the scope of the whole initiative,<br />
we made decide to repeat the "Do" and "Check" phases, incorporating our additional improvements.<br />
<br><br />
<br><br />
Once you are finally satisfied that the costs would outweigh the benefits of repeating the Do-Check sub-cycle any more,<br />
you can move on to the final phase.<br />
<br><br />
<br><br />
<z3>Act</z3><br />
<br><br />
<br><br />
After implement our solution we generated part of a continuous improvement initiative, we made a loop back to the Plan Phase,<br />
and seek out further areas for improvement.<br />
<br> <br />
<br><br />
<z2>Material</z2><br />
<br><br />
<br><br />
Two different materials were carefully chosen for our final product. At first, the outer <br />
compartment should be resistant enough to support the bacteria inside the sticker and give <br />
resistance to the pressure that is applied by the customer. Its physical properties and characteristics<br />
should not be affected when too much pressure or deformation is applied on the sticker. It should be<br />
light and small, to ensure the capsule is easily placed in meat package, for instance.<br />
Second, the case should be made from a light material and the surface should be flat to provide grip <br />
and avoid sharp edges or any other risks for the user.<br />
<br><br />
<br><br />
Taking into account all these characteristics, we considered three materials for the sticker: <br />
polyetheen (sandwich bag), Polyvinyl chloride (cling film) and Polymethylpentene (PMP) als commonly<br />
known as TPX®. We chose TPX® as the most suitable material for the outer layer of the sticker, because <br />
this material fulfills all the requirements mentioned above. <br />
<br> <br />
<br><br />
In addition, we searched for another material needed for the inner compartment of the sticker, <br />
that separates the growth medium from the spores untill the consumer wants to use it. At first, <br />
polyvinyl chloride turned out to be too weak for the outer layer of the sticker, because it is easily broken.<br />
However, these properties are very suitable for the needed breakable inner compartment of the sticker.<br />
Very little pressure is needed to break the inner compartment, so it will be easy to start <br />
the Food Warden system when the consumer needs it.<br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.<br><br />
2. H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)<br><br />
3. Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.<br><br />
4. FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).<br><br />
5. Mathiowetz V, Kashman N, Volland G, Weber K, Dowe M, Rogers S (February 1985). "Grip and pinch strength: normative data for adults". Arch Phys Med Rehabil 66 (2): 69–74. PMID 3970660.<br><br />
</p><br />
<p><br />
<br><br />
<br><br />
<z2>Tools and supplies</z2><br />
<ol class="ref"><br />
<li>Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.</li><br />
<li>Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.</li><br />
<li>Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.</li><br />
<li>B-D Plastipak 21G 1<sup>1/2</sup> 40/8 NR2 2ml to fill up the sticker.</li> <br />
<li>Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.</li> <br />
<li>TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.</li><br />
<li>Luria Broth Brand: Lennox.</li><br />
</ol><br />
<br><br />
</p><br />
<p><br />
<z2>Prototype</z2><br />
<br><br />
<br> <br />
The prototype of the inner compartment is made from cling film with a side affix of 1 mm thickness. <br />
The subsequent created inner compartment was filled with sterilized Luria Broth (LB). In the outer layer of the sticker, <br />
made of TPX®, was filled with the cling film package and the bacterial spores. An important feature that we had to take into <br />
account was the oxygen exchange between the TPX® and the LB growth medium. If this does not occur extensively, <br />
the bacteria will not grow at their optimal rate. Therefore, we performed several experiments to calculate the minimum <br />
oxygen exchange. We used closed flasks containing 50ml medium and 84ml air to test the bacterial growth at 37 degrees Celsius. <br />
The 50 ml medium in the flasks had a diameter with an average of 6 cm where 84ml of air average of 21% oxygen at a starting point <br />
is applied on the medium in the flask. That makes 17.64ml of total oxygen that is needed for the growth of 50ml medium. <br />
From the TPX® characteristics and mathematical calculations we made the following graph.<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_ID_20120924_Foodwarden_sticker_surface_graph.png" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
This graph represent the minimum surface cm<sup>2</sup> is needed per ml. The green line is TPX® 50 um thickness and the red line is<br />
25um thickness. The x-axis is the volume of ml Luria Broth containing the Food Warden bacterium. <br />
The y-axis is the required surface area (cubic cm) per mL at 37 degrees Celsius.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<p><br />
<br><br />
<z2>Test</z2><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_ID_20120924_Sticker_diffrent_volumes_COrr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
We want to check if each different volume is equally easy to break.<br />
</p><br />
<td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_ID_20120924_Sticker_diffrent_volumes_before_growCorr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
All different volumes just after breakage, the LB broth has no traces of bacterial growth.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the medium with the Food warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Groningen2012_ID_20120927_Sticker_before_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
No growth in the sticker when it was placed first in a bottle with rotten meat (probably due to oxygen depletion), <br />
therefore we placed the sticker in a bottle with fresh meat<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_ID_20120925_sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Bottle of rotten meat connected to the microarray set up to ensure the oxygen is pumped around<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<z2>Results</z2><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/rlaCpIiV-24"></iframe><br />
<br><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
In the movie we see how easy the sticker is to break. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/Groningen2012_ID_20120924_Sticker_diffrent_volumes_grow_Corr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
We tested different kind of volumes to verify the calculations in the surface area plot.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the medium with the Food Warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/49/Groningen2012_ID_20120927_Sticker_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
First picture of the <i>Bacillus</i> producing the purple AmilCP pigment IN the sticker and not in a bottle of the microarray set up.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food Warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/Groningen2012_ID_20120927_Sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
Are these bacteria going to produce pigment before or after the wikifreeze. That´s the question!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
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<div class="ctd"><br />
<z1 >The sticker</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
Not everyone likes free living, engineered bacteria next to their food. But in order to make our Food Warden<br />
system work properly, our bacteria should be able to react to the volatiles coming off the spoiling meat while it<br />
is located in, for instance, a meat package. We had a lot of discussions about how to achieve a very safe and <br />
easy-in-use solution, and this is what we came up with:<br />
<br><br />
<br> <br />
<i>Bacillus subtilis</i> would be an ideal candidate as a chassis for our genetically engineered construct because it <br />
has the ability to form endospores, a kind of dormant state that they use for survival. They can survive high levels<br />
of heat (>100 °C), drying, radiation, and many damaging chemicals and simply be brought back 'to life' under the influence<br />
of sufficient nutrients and water. This restorative process is germination. For the information, <br />
take a look <a class="inlink" href="http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=69">here</a> and <br />
<a class="inlink" href="http://www.pearsonhighered.com/pearsonhigheredus/educator/product/products_detail.page?isbn=0132324601">here</a>. <br />
Because of this ability to go dormant, our bacterium can be stored and activated when it is needed!<br />
<br><br />
<br> <br />
However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a <br />
containment device to store and activate the spores at the right time. For clarity: our team keeps calling it 'the sticker' all the time.<br />
The sticker consists of two nested compartments. The inner compartment contains a calibrated amount of nutrients, <br />
while the outer semi-permeable capsule contains the spores of our engineered strain. Breaking the barrier between the two compartments <br />
allows germination and growth of <i>Bacillus subtilis</i> cells. <br />
<br><br><br />
Now the properties of the material we use for our sticker come into play. The polymer we used is TPX®, also<br />
called polymethylpentene. This polymer is available as thin, transparent sheets. The advantage is that it is <br />
relatively cheap, strong, and capable of letting through volatiles. The radius of the pores in TPX® is between <br />
1 nm and 10 nm, which is at least 50x larger than the average badmeat-volatile, but still small enough to keep liquid <br />
and bacteria or spores in (see the figure below). More detailed information about the sticker design and its experiments, are stated below. <br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Siebring J. 2012 (unpublished)<br />
</p><br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/RR_20120825_tpx.PNG" width="300"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Comparison of the size of the TPX® pores, volatiles and <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
<z2>Design requirements</z2><br />
<br><br />
<br><br />
<z3>Material</z3><br />
<br><br />
<br><br />
The material should not break easily and should resist a human grip strength with a minimum of 40 pounds pinch and pressure. <br><br />
The product should be made of a material that is light and easy to handle. <br><br />
The material should be durable and inexpensive.<br> <br />
The volatiles and oxygen should penetrate through the material, while the spores, bacteria and liquid should stay inside.<br><br />
The material should fit in a meat package.<br><br />
The material should not be toxic or become toxic for the bacteria <br><br />
The material should be able to cope with a temperature of at least 125 degrees Celsius.<br />
<br><br />
<br><br />
<z3>Measurements</z3><br />
<br><br />
<br><br />
We want a visible feedback system for the human eye that should easy to understand for the consumer. <br><br />
The visible feedback should not degrade over time.<br />
<br><br />
<br><br />
<z3>Appearance</z3><br />
<br><br />
<br><br />
Product should be have attractive color(s) and recognizable shape.<br />
<br> <br />
<br><br />
<z3>Safety</z3><br />
<br><br />
<br><br />
The bacteria should not escape from the sticker nor harm the environment or the costumer. <br />
Therefore the product should provide adequate support and be reliable.<br />
<br><br />
<br><br />
<z3>Customer comfort</z3><br />
<br><br />
<br><br />
Easy to use the visible feedback device. <br />
<br><br />
The product may provide different degrees of strength depending on the development of the consumer strength. <br />
<br><br />
<br><br />
<z2>Development design</z2><br />
<br><br />
<br><br />
For the development of the sticker, the PDCA (Plan Do Check Act) strategy was used to develop an ultimate <br />
device that keeps track of the needs of the customer and the environment in the project design.<br />
<br><br />
<br> <br />
The following questions where raised: <br />
<br><br />
<br><br />
Who is your external customer?<br />
<br><br />
The customer who is against food spoilage and customers that are willing to buy it.<br />
<br><br />
<br> <br />
What value do you want to deliver to that customer?<br />
<br><br />
A replacement for the "use by date and sell by date" system. We want to develop a new product that <br />
indicates when meat starts to spoil before the costumer can notice this by eye, by smell or by taste.<br />
<br> <br />
<br><br />
Who, in you iGEM group, delivers that value?<br />
<br><br />
The design engineers and the biologists of iGEM Groningen 2012.<br />
<br><br />
<br><br />
How do they deliver that value?<br><br />
By doing research and creating a new indicator to predict when meat starts to spoil.<br />
<br><br />
<br><br />
<z3>Plan</z3><br />
<br><br />
<br><br />
Our first plan is to identify exactly what we have to do to make a sticker. <br />
The sticker should be the same as the use of a glow-in-the-dark stick, that you first have to<br />
break before it gives off a bright chemical light. Everyone should understand how <br />
it works. The indication color might be made with the same colors as a traffic light, to <br />
indicate when the meat is fresh, a bit spoiled or really spoiled. During our second case, <br />
we want to produce an easily activated sticker. In the third part of the plan, the outer sticker should be strong enough, <br />
let volatiles go through the outer layer of the sticker, and not any bacteria or liquid should go<br />
out of the outer layer of the sticker.<br />
<br><br />
<br><br />
</p><br />
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<br><br />
<br><br />
<br> <br />
<p><br />
<z3>Do</z3><br />
<br><br />
<br><br />
The following solution was chosen:<br />
Build a transparent plastic bag with inside a smaller compartment, where the medium inside is visible for the user when the color changes.<br />
Make the inner compartment of a weaker material where the sides break when a pinch force is applied.<br />
<br><br />
<br><br />
The following pilots where made:<br><br />
<br><br />
1. Make sticker user-friendly concerning the breakage of the inner compartment.<br><br />
2. Test if the bacteria grow in the sticker.<Br><br />
3. Test if the medium with the Food Warden bacteria will product a pigment in the sticker. <br />
<br><br />
<br><br />
<z3>Check</z3><br />
<br><br />
<br><br />
Depending on the success of the pilots, the number of areas for improvement you have identified, and the scope of the whole initiative,<br />
we made decide to repeat the "Do" and "Check" phases, incorporating our additional improvements.<br />
<br><br />
<br><br />
Once you are finally satisfied that the costs would outweigh the benefits of repeating the Do-Check sub-cycle any more,<br />
you can move on to the final phase.<br />
<br><br />
<br><br />
<z3>Act</z3><br />
<br><br />
<br><br />
After implement our solution we generated part of a continuous improvement initiative, we made a loop back to the Plan Phase,<br />
and seek out further areas for improvement.<br />
<br> <br />
<br><br />
<z2>Material</z2><br />
<br><br />
<br><br />
Two different materials were carefully chosen for our final product. At first, the outer <br />
compartment should be resistant enough to support the bacteria inside the sticker and give <br />
resistance to the pressure that is applied by the customer. Its physical properties and characteristics<br />
should not be affected when too much pressure or deformation is applied on the sticker. It should be<br />
light and small, to ensure the capsule is easily placed in meat package, for instance.<br />
Second, the case should be made from a light material and the surface should be flat to provide grip <br />
and avoid sharp edges or any other risks for the user.<br />
<br><br />
<br><br />
Taking into account all these characteristics, we considered three materials for the sticker: <br />
polyetheen (sandwich bag), Polyvinyl chloride (cling film) and Polymethylpentene (PMP) als commonly<br />
known as TPX®. We chose TPX® as the most suitable material for the outer layer of the sticker, because <br />
this material fulfills all the requirements mentioned above. <br />
<br> <br />
<br><br />
In addition, we searched for another material needed for the inner compartment of the sticker, <br />
that separates the growth medium from the spores untill the consumer wants to use it. At first, <br />
polyvinyl chloride turned out to be too weak for the outer layer of the sticker, because it is easily broken.<br />
However, these properties are very suitable for the needed breakable inner compartment of the sticker.<br />
Very little pressure is needed to break the inner compartment, so it will be easy to start <br />
the Food Warden system when the consumer needs it.<br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.<br><br />
2. H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)<br><br />
3. Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.<br><br />
4. FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).<br><br />
5. Mathiowetz V, Kashman N, Volland G, Weber K, Dowe M, Rogers S (February 1985). "Grip and pinch strength: normative data for adults". Arch Phys Med Rehabil 66 (2): 69–74. PMID 3970660.<br><br />
</p><br />
<p><br />
<br><br />
<br><br />
<z2>Tools and supplies</z2><br />
<ol class="ref"><br />
<li>Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.</li><br />
<li>Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.</li><br />
<li>Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.</li><br />
<li>B-D Plastipak 21G 1<sup>1/2</sup> 40/8 NR2 2ml to fill up the sticker.</li> <br />
<li>Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.</li> <br />
<li>TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.</li><br />
<li>Luria Broth Brand: Lennox.</li><br />
</ol><br />
<br><br />
</p><br />
<p><br />
<z2>Prototype</z2><br />
<br><br />
<br> <br />
The prototype of the inner compartment is made from cling film with a side affix of 1 mm thickness. <br />
The subsequent created inner compartment was filled with sterilized Luria Broth (LB). In the outer layer of the sticker, <br />
made of TPX®, was filled with the cling film package and the bacterial spores. An important feature that we had to take into <br />
account was the oxygen exchange between the TPX® and the LB growth medium. If this does not occur extensively, <br />
the bacteria will not grow at their optimal rate. Therefore, we performed several experiments to calculate the minimum <br />
oxygen exchange. We used closed flasks containing 50ml medium and 84ml air to test the bacterial growth at 37 degrees Celsius. <br />
The 50 ml medium in the flasks had a diameter with an average of 6 cm where 84ml of air average of 21% oxygen at a starting point <br />
is applied on the medium in the flask. That makes 17.64ml of total oxygen that is needed for the growth of 50ml medium. <br />
From the TPX® characteristics and mathematical calculations we made the following graph.<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_ID_20120924_Foodwarden_sticker_surface_graph.png" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
This graph represent the minimum surface cm<sup>2</sup> is needed per ml. The green line is TPX® 50 um thickness and the red line is<br />
25um thickness. The x-axis is the volume of ml Luria Broth containing the Food Warden bacterium. <br />
The y-axis is the required surface area (cubic cm) per mL at 37 degrees Celsius.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<p><br />
<br><br />
<z2>Test</z2><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_ID_20120924_Sticker_diffrent_volumes_COrr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
We want to check if each different volume is equally easy to break.<br />
</p><br />
<td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_ID_20120924_Sticker_diffrent_volumes_before_growCorr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
All different volumes just after breakage, the LB broth has no traces of bacterial growth.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the medium with the Food warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Groningen2012_ID_20120927_Sticker_before_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
No growth in the sticker when it was placed first in a bottle with rotten meat (probably due to oxygen depletion), <br />
therefore we placed the sticker in a bottle with fresh meat<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra test Test if the medium with the Food warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_ID_20120925_sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Bottle of rotten meat connected to the microarray set up to ensure the oxygen is pumped around<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<z2>Results</z2><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/rlaCpIiV-24"></iframe><br />
<br><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
In the movie we see how easy the sticker is to break. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/Groningen2012_ID_20120924_Sticker_diffrent_volumes_grow_Corr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
We tested different kind of volumes to verify the calculations in the surface area plot.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Test if the medium with the Food Warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/49/Groningen2012_ID_20120927_Sticker_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
First picture of the <i>Bacillus</i> producing the purple AmilCP pigment IN the sticker and not in a bottle of the microarray set up.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra test Test if the medium with the Food warden bacteria will product a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/Groningen2012_ID_20120927_Sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
Are these bacteria going to produce pigment before or after the wikifreeze. That´s the question!<br />
</p><br />
</td><br />
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<div class="expand-down"><br />
<ul id="navigation" ><br />
<li class="sub" id="menuhome"><br />
<br />
<a href="https://2012.igem.org/Team:Groningen" title="Home" rel="nofollow"><br />
<img src="https://static.igem.org/mediawiki/2012/6/61/Groningen2012_ID_20120808_Menu_Home.png" alt="Home" title="Home" /><br />
<span>home</span><br />
</a><br />
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<div class="cogbig"><br />
<img id="cogbighome" src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="80" height="80"><br />
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<br />
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<div class="cogbig"> <br />
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</div><br />
<a href="https://2012.igem.org/Team:Groningen/Team" title="Team" rel="nofollow" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/Groningen2012_ID_20120808_Menu_Team.png" alt="team" title="Team" /><br />
<span>team</span><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Groningen/Team">about us</a></li><br />
<li><a href="https://igem.org/Team.cgi?id=818" TARGET="_blank">official profile</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Gallery">gallery</a></li><br />
<br />
<li><a href="https://2012.igem.org/Team:Groningen/Attributions">attributions</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/acknowledgements">acknowledgements</a></li><br />
<br />
<li><a href="https://2012.igem.org/Team:Groningen/Contact">contact</a></li><br />
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<div class="cogbig"><br />
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<a href="https://2012.igem.org/Team:Groningen/Project" title="Project" rel="nofollow" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/19/Groningen2012_ID_20120808_Menu_Project.png" alt="Project" title="Project" /><br />
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<ul><br />
<li><a href="https://2012.igem.org/Team:Groningen/Project">abstract</a></li> <li><a href="https://2012.igem.org/Team:Groningen/Data_page">data page</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Sticker">sticker</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/volatiles">volatiles</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Sensor">sensor</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/pigmentproduction">pigment</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Construct">construct</a></li><br />
<li><a href="https://2012.igem.org/Team:Groninge/Kill_Switch">kill switch</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Modeling">modeling</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Notebook">notebook</a></li> <br />
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<div class="cogbig"><br />
<img id="cogbigparts" src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="80" height="80"><br />
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<a href="https://2012.igem.org/Team:Groningen/OurBiobrick" title="Parts" rel="nofollow" ><br />
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<span>parts</span><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Groningen/OurBiobrick">our biobricks</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/parts_fail">improvements</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/in_development">in development</a></li><br />
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<span>safety</span><br />
<ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Groningen/Safety">general safety</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/10com">in the lab</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/publicsafety">public safety</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/environment">environment</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/foodsafety">food safety</a></li><br />
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<div class="cogbig"><br />
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<a href="https://2012.igem.org/Team:Groningen/overview" title="Public Relation" rel="nofollow" ><br />
<img src="https://static.igem.org/mediawiki/2012/6/6f/Groningen2012_ID_20120808_Menu_Public_Relations.png" alt="Public Relation" title="Public Relation" /><br />
<span>human<br>practice</span><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Groningen/overview">overview</a></li> <br />
<li><a href="https://2012.igem.org/Team:Groningen/summer_school">summerschool</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative">stop food waste</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/market_research">market research</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Festivals">festivals</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/symposia">presentations</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/international_cooperation">collaboration</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/Media_coverage">media coverage</a></li><br />
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<div class="cogbig"><br />
<img id="cogbigsponsors" src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="80" height="80"><br />
</div><br />
<a href="https://2012.igem.org/Team:Groningen/Sponsors" title="Sponsors" rel="nofollow" ><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Groningen2012_ID_20120808_Menu_Sponsors.png" alt="Sponsors" title="Sponsors" /><br />
<span>sponsors</span><br />
<ul> <br />
<li><a href="https://2012.igem.org/Team:Groningen/our_sponsors">our sponsors</a></li><br />
<li><a href="https://2012.igem.org/Team:Groningen/sponsor_levels">sponsorship levels</a></li><br />
</ul> <br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/our_sponsorsTeam:Groningen/our sponsors2012-10-26T19:45:17Z<p>Jparrish: </p>
<hr />
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</style><br />
</head><br />
<br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Our Sponsors</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
<z2>Platinum level</z2> (investment of >3.000€)<br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<table class="tablewhite"><br />
<tr><br />
<td class="platinumcell" colspan="2"><br />
<a href="http://www.rug.nl/fmns-research/gbb/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_JP_20120813_Gratio_GBB.jpg" width="200px"/></a><br />
</td><br />
</tr><br />
<tr><br />
<td class="platinumcell"><br />
<a href="http://www.rug.nl/corporate/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/2c/Groningen2012_JP_20120813_Gratio_RuG.jpg" width="300px"/></a><br />
</td><br />
<td class="platinumcell"><br />
<a href="http://nbv.kncv.nl/stichting-biotechnologie-nederland-%28sbn%29.7252.lynkx"><img src="https://static.igem.org/mediawiki/2012/5/5f/Groningen2012_JP_20120813_Gratio_SBN.jpg" width="300px"/></a><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Gold level</z2> (investment of >1.000€)<br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<table class="tablewhite"><br />
<tr><br />
<td class="goldcell"><br />
<a href="http://www.erasynbio.eu/project" target="_blank"><img src="https://static.igem.org/mediawiki/2012/7/7f/Groningen2012_JP_20121022_SynBio.jpg" width="250px"/></a><br />
</td><br />
<td class="goldcell"><br />
<a href="http://www.rug.nl/fmns-research/csb/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_JP_20121022_CSB.jpg" width="250px"/></a><br />
</td><br />
<td class="goldcell"><br />
<a href="http://www.maxgruber-foundation.nl/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/f/fe/Groningen2012_JP_20121022_Max.jpg" width="250px"/></a><br />
</td><br />
</tr><br />
</table><br />
<table class="tablewhite"><br />
<tr><br />
<td class="goldcell"><br />
<a href="http://www.dsm.com/nl_NL/dsmnl/public/home/pages/home.jsp" target="_blank"><img src="https://static.igem.org/mediawiki/2012/7/78/Groningen2012_JP_20120813_Gratio_DSM.jpg" width="250px"/></a><br />
</td><br />
<td class="goldcell"><br />
<a href="http://www.rug.nl/fmns-research/zernike/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_JP_20120924_Gratio_ZIAM.jpg" width="250px"/></a><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Silver level</z2> (investment of >500€)<br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<table class="tablewhite"><br />
<tr><br />
<td class="silvercell"><br />
<a href="http://molgen.biol.rug.nl/molgen/index.php" target="_blank"><img src="https://static.igem.org/mediawiki/2012/a/a2/Groningen2012_JP_20120813_Gratio_MolGen.jpg" width="200px"/></a><br />
</td><br />
<td class="silvercell"><br />
<a href="http://www.rug.nl/fmns-research/alice/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/7/7c/Groningen2012_JP_20120924_Gratio_ALICE.jpg" width="200px"/></a><br />
</td><br />
<td class="silvercell"><br />
<a href="http://www.sonac.biz/nl/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20121022_SONAC.jpg" width="200px"/></a><br />
</td><br />
</tr><br />
</table><br />
<table class="tablewhite"><br />
<tr><br />
<td class="silvercell"><br />
<a href="http://www.rug.nl/sciencelinx/index" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/88/Groningen2012_JP_20120924_Gratio_SLinx.jpg" width="200px"/></a><br />
</td><br />
<td class="silvercell"><br />
<a href="http://www.sigmaaldrich.com/nederland.html" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/d0/Groningen2012_JP_20120813_Gratio_Sigma.jpg" width="200px"/></a><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Bronze level</z2> <br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<table class="tablewhite"><br />
<tr><br />
<td class="bronzecell"><br />
<a href="http://www.thermofisher.com/global/en/home.asp" target="_blank"><img src="https://static.igem.org/mediawiki/2012/6/60/Groningen2012_JP_20120813_Gratio_ThermoFisher.jpg" width="150px"/></a><br />
</td><br />
<td class="bronzecell"><br />
<a href="http://www.infors-ht.com/index.php/en/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20120924_Gratio_Infors.jpg" width="150px"/></a><br />
</td><br />
<td class="bronzecell"><br />
<a href="http://eu.idtdna.com/site" target="_blank"><img src="https://static.igem.org/mediawiki/2012/1/1a/Groningen2012_JP_20120813_Gratio_IDT.jpg" width="150px"/></a><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>IndieGogo</z2><br />
<br><br />
<br><br />
A thanks to those private citizens who donated through the crowdfunding platform.<br />
<br><br />
Thank you Henky Kuntoro and Marleen Feteris.<br />
</p><br />
<br><br />
<br><br />
<p><br />
<z2>Sponsored Material</z2><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Groningen_RR_20120810_sigma.jpg" width="500" ><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="nomargin"><br />
Thanks to Sigma-Aldrich for the free materials!!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br><br />
<p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/af/Groningen_RR20120810_thermoscientific.jpg" width="500" ><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="nomargin"><br />
Thanks to ThermoFisher Scientific for their discount on enzymes and kits!!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
</body><br />
</html></div>Jparrishhttp://2012.igem.org/Template:SponsorsGroningen2012Template:SponsorsGroningen20122012-10-26T19:34:21Z<p>Jparrish: </p>
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<h1>Our sponsors:</h1><br />
<div class="rowlevel"><br />
<a href="http://www.rug.nl/corporate/index" target="_blank"><img class="platinum" src="https://static.igem.org/mediawiki/2012/2/2c/Groningen2012_JP_20120813_Gratio_RuG.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/gbb/index" target="_blank"><img class="platinumsmall" src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_JP_20120813_Gratio_GBB.jpg"/></a><br />
<a href="http://nbv.kncv.nl/stichting-biotechnologie-nederland-%28sbn%29.7252.lynkx"><img class="platinum" src="https://static.igem.org/mediawiki/2012/5/5f/Groningen2012_JP_20120813_Gratio_SBN.jpg"/></a><br />
</div><br />
<div class="rowlevel"><br />
<a href="http://www.dsm.com/nl_NL/dsmnl/public/home/pages/home.jsp" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/7/78/Groningen2012_JP_20120813_Gratio_DSM.jpg"/></a><br />
<a href="http://www.erasynbio.eu/project" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/7/7f/Groningen2012_JP_20121022_SynBio.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/csb/index" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_JP_20121022_CSB.jpg"/></a><br />
<a href="http://www.maxgruber-foundation.nl/" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/f/fe/Groningen2012_JP_20121022_Max.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/zernike/index" target="_blank"><img class="gold" height="70px" src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_JP_20120924_Gratio_ZIAM.jpg"/></a><br />
</div><br />
<div class="rowlevel"><br />
<a href="http://www.rug.nl/sciencelinx/index" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/8/88/Groningen2012_JP_20120924_Gratio_SLinx.jpg"/></a><br />
<a href="http://molgen.biol.rug.nl/molgen/index.php" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/a/a2/Groningen2012_JP_20120813_Gratio_MolGen.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/alice/index" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/7/7c/Groningen2012_JP_20120924_Gratio_ALICE.jpg"/></a><br />
<a href="http://www.sonac.biz/nl/" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20121022_SONAC.jpg"/></a><br />
<a href="http://www.sigmaaldrich.com/nederland.html" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/d/d0/Groningen2012_JP_20120813_Gratio_Sigma.jpg"/></a><br />
</div><br />
<div class="rowlevel"><br />
<a href="http://www.thermofisher.com/global/en/home.asp" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/6/60/Groningen2012_JP_20120813_Gratio_ThermoFisher.jpg"/></a><br />
<a href="http://www.infors-ht.com/index.php/en/" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20120924_Gratio_Infors.jpg"/></a><br />
<a href="http://eu.idtdna.com/site" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/1/1a/Groningen2012_JP_20120813_Gratio_IDT.jpg"/></a><br />
</div><br />
</body><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/DesignTestTeam:Groningen/DesignTest2012-10-26T19:28:43Z<p>Jparrish: </p>
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<h1>Our sponsors:</h1><br />
<div class="rowlevel"><br />
<a href="http://www.rug.nl/corporate/index" target="_blank"><img class="platinum" src="https://static.igem.org/mediawiki/2012/2/2c/Groningen2012_JP_20120813_Gratio_RuG.jpg"/></a><br />
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<a href="http://nbv.kncv.nl/stichting-biotechnologie-nederland-%28sbn%29.7252.lynkx"><img class="platinum" src="https://static.igem.org/mediawiki/2012/5/5f/Groningen2012_JP_20120813_Gratio_SBN.jpg"/></a><br />
</div><br />
<div class="rowlevel"><br />
<a href="http://www.erasynbio.eu/project" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/7/7f/Groningen2012_JP_20121022_SynBio.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/csb/index" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_JP_20121022_CSB.jpg"/></a><br />
<a href="http://www.dsm.com/nl_NL/dsmnl/public/home/pages/home.jsp" target="_blank"><img class="gold" src="https://static.igem.org/mediawiki/2012/7/78/Groningen2012_JP_20120813_Gratio_DSM.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/zernike/index" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_JP_20120924_Gratio_ZIAM.jpg"/></a><br />
<a href="http://www.maxgruber-foundation.nl/" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/f/fe/Groningen2012_JP_20121022_Max.jpg"/></a><br />
<br />
</div><br />
<div class="rowlevel"><br />
<a href="http://molgen.biol.rug.nl/molgen/index.php" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/a/a2/Groningen2012_JP_20120813_Gratio_MolGen.jpg"/></a><br />
<a href="http://www.rug.nl/fmns-research/alice/index" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/7/7c/Groningen2012_JP_20120924_Gratio_ALICE.jpg"/></a><br />
<a href="http://www.sonac.biz/nl/" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20121022_SONAC.jpg"/></a><br />
<a href="http://www.rug.nl/sciencelinx/index" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/8/88/Groningen2012_JP_20120924_Gratio_SLinx.jpg"/></a><br />
<a href="http://www.sigmaaldrich.com/nederland.html" target="_blank"><img class="silver" src="https://static.igem.org/mediawiki/2012/d/d0/Groningen2012_JP_20120813_Gratio_Sigma.jpg"/></a><br />
</div><br />
<div class="rowlevel"><br />
<a href="http://www.thermofisher.com/global/en/home.asp" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/6/60/Groningen2012_JP_20120813_Gratio_ThermoFisher.jpg"/></a><br />
<a href="http://www.infors-ht.com/index.php/en/" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_JP_20120924_Gratio_Infors.jpg"/></a><br />
<a href="http://eu.idtdna.com/site" target="_blank"><img class="bronze" src="https://static.igem.org/mediawiki/2012/1/1a/Groningen2012_JP_20120813_Gratio_IDT.jpg"/></a><br />
</div><br />
</body><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-10-26T19:21:30Z<p>Jparrish: </p>
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</head><br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Volatiles</z1><br />
</div><br />
</div><br />
<p><br />
<z2>When is meat rotten?</z2><br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/RR_20120807_TAMCpic.jpg" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
To make a rotting-meat sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union <br />
(2006, source <a class="inlink" href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target="_blank">(in Dutch)</a>)<br />
and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram of meat. <br />
See our <a class="inlink" href="https://2012.igem.org/Team:Groningen/foodsafety">food safety page</a> for more in depth answer to this question. <br />
Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of<br />
meat is often bought in large amounts with leftovers stored in the fridge, making it the ideal candidate for our Food Warden system.<br />
Minced meat is also easy to handle when it is placed in a jar, simplifying lab work. Most importantly, as a meat lover it is hard to sacrifice a<br />
very nice expensive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the <br />
TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br />
<br><br />
<br><br />
</p><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG" width="300"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Results of TAMC counting. TAMC (colony forming units/gram meat, y axis) of meat incubated at room temperature for indicated time (x axis). Red area indicates the critical <br />
values where the meat is not allowed to be distributed for consumption according to the EU.<br />
<br><br />
<br><br />
</p> <br />
<p><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, <br />
we humans can smell bad meat rather well. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: <br />
it turned greyer. <br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Smelling test. Minced meat was left at room temperature for 6 hours. The “nastiness” of the meat smell according to the tester was recorded.</p><br />
<br><br />
</p><br />
<p><br />
Now that we defined and smelled rotten meat, we want to know what the volatiles are. The rotting of the meat is caused by bacteria proliferating in the meat. <br />
These bacteria produce a lot of volatiles and we can only smell a few. To form a complete picture of the aromatic and non-aromatic volatiles we analyzed the <br />
rotten meat volatiles with GC-MS.<br />
<br><br />
<br><br />
<z2>Volatiles from rotten meat</z2><br />
<br><br />
<br><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that <br />
the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor" target="_blank">sensors</a>, but we were surprised by what we found…<br />
<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the <br />
substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify <br />
from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the <br />
GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity <br />
of the volatiles present. <br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="350"><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_EH_20120727_P7270580.JPG" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center" width="350px"><br />
<p class="captionnomargin"><br />
Arjan and Tom at work with the GC-MS.<br />
</p><br />
</td><br />
<td align="center" width="350px"><br />
<p class="captionnomargin"><br />
Sample bottles with rotten minced meat.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/7/73/Groningen2012_EH_20120727_P7270583.JPG" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Introduction to the GC-MS technique</z2><br />
<br><br />
<br><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, <br />
in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase <br />
and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is <br />
traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier<br />
is based on the boiling point of the substrate. Each substrate has an unique amount of interactions with the carrier and thus a different <br />
amount of time is needed to pass through the column. Based on this principle, our rotten-meat volatiles are separated and later identified<br />
with mass spectrometry.<br />
<br><br />
<br><br />
We used the headspace method to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high <br />
temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was controlled <br />
as in microarray experiment for the identification of pBAD-meat<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor" target="_blank">sensors</a>.<br />
<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There are a broad range of columns commercially available, and all are capable of separating <br />
substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at RUG utilize these columns,<br />
but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotten <br />
meat and unfortunately we did not have the budget to buy more specific columns.<br />
<br><br />
<br><br />
HP-5:<br><br />
Dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
Manufacturer: Agilent<br> <br />
Column number: 19091J-433<br><br />
Stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
Dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
Manufacturer: Agilent<br> <br />
Column number: 19091Z-433<br><br />
Stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds<br />
based on their differences in mass-to-charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that<br />
the separate substrates in the machine will be bombarded with electrons in a vaccuum. Because of this, they will receive a charge and are most likely <br />
to break into pieces. These flying bits and pieces of substrates, each with their own charge, will be prevented from crashing into the wall by the<br />
quadruple poles. One can adjust the changing charge of the poles and thereby choose the range of spectrum of the identifiable compounds with their <br />
different mass-to-charge ratios.<br />
<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules (which might cause redundant mass charge ratios), <br />
and how small the differences in molecule structure are (like isomers) that are difficult to separate.<br />
<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra. Unfortunately, the concentration of volatiles was probably too low in the extracted <br />
sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles.<br />
In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine<br />
as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature.<br />
Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
<br><br />
But also this did not work out, because we got blank spectra again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better<br />
than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not give up, though.<br />
<br><br />
<br><br />
<z2>Liquid injection with organic solvents</z2><br />
<br><br />
<br><br />
The first measurements with only volatiles gave no reliable results. Therefore, we decided to dissolve the rotten meat into organic solvents to ensure that<br />
more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot<br />
for more than a day at room temperature before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent<br />
for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent;<br />
we needed to study this thoroughly, and to cover all the polar and non-polar volatiles, we used different organic solvents.<br />
<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat. In order to prevent damage to the columns <br />
we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br />
<br><br />
<br> <br />
<z2>Results</z2><br />
<br><br />
<br><br />
Finally, this method produced interesting results from both the HP-1 and HP-5 column spectra. After the library search; only compounds <br />
with a quality over 80% were considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra<br />
of fresh meat and the solvent blank. Subtracting the solvent blank removes the background noise, while subtracting the fresh meat highlights those spectra <br />
up- and downregulated in rotten-meat volatiles. This approach identified the following compounds in rotten meat, <br />
with a quality that matches with approximately 80% to the reference library.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width=350 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" class="preview" width=600 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width=350 /><br />
<img src="https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" class="preview" width=600 /><br />
</a><br />
</li><br />
</ul><br />
<p><br />
<br><br />
We discussed our results with Prof. Dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 <br />
fatty acid, which is expected not to come out of the columns easily. This substrate is also found on both columns and with different solvents.<br />
<br><br />
<br><br />
He also noted that the overall reference quality was low because results with less than 80% quality are considered as unreliable. This is probably caused by compounds<br />
that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br />
<br><br />
<br> <br />
Some of the compounds found contain a fluoro group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded<br />
these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrates are also not likely to be correct. An explanation for this <br />
lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to <br />
known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer ‘blindly’ decides what the best fitting substrate spectra is. <br />
These fits can be incorrect, so we should take care when extracting conclusions from these results.<br />
<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other <br />
interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br />
<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra are <br />
compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform <br />
more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, not the major part. We decided to spend our time on other research <br />
although Prof. Minnaard suggested that we should do another microarray experiment using these compounds instead of the rotten meat.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxM"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxR"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<p class="caption"><br />
GC-MS spectra using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxM"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxR"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<p class="caption"><br />
GC-MS spectra using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Future plans</z2><br />
<br><br />
<br><br />
If time allows, we will try to set up an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. <br />
Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the<br />
growth of <i>B. subtilis</i> with our construct.<br />
<br><br />
<br><br />
<z2>Reference and acknowledgement</z2><br />
<br><br />
<br><br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry <br />
for her valuable help during the measurements and her explanations about the technique.<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-10-26T19:20:33Z<p>Jparrish: </p>
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<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Volatiles</z1><br />
</div><br />
</div><br />
<p><br />
<z2>When is meat rotten?</z2><br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/RR_20120807_TAMCpic.jpg" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
To make a rotting-meat sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union <br />
(2006, source <a class="inlink" href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target="_blank">(in Dutch)</a>)<br />
and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram of meat. <br />
See our <a class="inlink" href="https://2012.igem.org/Team:Groningen/foodsafety">food safety page</a> for more in depth answer to this question. <br />
Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of<br />
meat is often bought in large amounts with leftovers stored in the fridge, making it the ideal candidate for our Food Warden system.<br />
Minced meat is also easy to handle when it is placed in a jar, simplifying lab work. Most importantly, as a meat lover it is hard to sacrifice a<br />
very nice expensive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the <br />
TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br />
<br><br />
<br><br />
</p><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG" width="300"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Results of TAMC counting. TAMC (colony forming units/gram meat, y axis) of meat incubated at room temperature for indicated time (x axis). Red area indicates the critical <br />
values where the meat is not allowed to be distributed for consumption according to the EU.<br />
<br><br />
<br><br />
</p> <br />
<p><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, <br />
we humans can smell bad meat rather well. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: <br />
it turned greyer. <br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Smelling test. Minced meat was left at room temperature for 6 hours. The “nastiness” of the meat smell according to the tester was recorded.</p><br />
<br><br />
</p><br />
<p><br />
Now that we defined and smelled rotten meat, we want to know what the volatiles are. The rotting of the meat is caused by bacteria proliferating in the meat. <br />
These bacteria produce a lot of volatiles and we can only smell a few. To form a complete picture of the aromatic and non-aromatic volatiles we analyzed the <br />
rotten meat volatiles with GC-MS.<br />
<br><br />
<br><br />
<z2>Volatiles from rotten meat</z2><br />
<br><br />
<br><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that <br />
the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor" target="_blank">sensors</a>, but we were surprised by what we found…<br />
<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the <br />
substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify <br />
from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the <br />
GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity <br />
of the volatiles present. <br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="350"><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_EH_20120727_P7270580.JPG" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td width="350px"><br />
<p class="captionnomargin"><br />
Arjan and Tom at work with the GC-MS.<br />
</p><br />
</td><br />
<td width="350px"><br />
<p class="captionnomargin"><br />
Sample bottles with rotten minced meat.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/7/73/Groningen2012_EH_20120727_P7270583.JPG" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Introduction to the GC-MS technique</z2><br />
<br><br />
<br><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, <br />
in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase <br />
and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is <br />
traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier<br />
is based on the boiling point of the substrate. Each substrate has an unique amount of interactions with the carrier and thus a different <br />
amount of time is needed to pass through the column. Based on this principle, our rotten-meat volatiles are separated and later identified<br />
with mass spectrometry.<br />
<br><br />
<br><br />
We used the headspace method to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high <br />
temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was controlled <br />
as in microarray experiment for the identification of pBAD-meat<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/Sensor" target="_blank">sensors</a>.<br />
<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There are a broad range of columns commercially available, and all are capable of separating <br />
substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at RUG utilize these columns,<br />
but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotten <br />
meat and unfortunately we did not have the budget to buy more specific columns.<br />
<br><br />
<br><br />
HP-5:<br><br />
Dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
Manufacturer: Agilent<br> <br />
Column number: 19091J-433<br><br />
Stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
Dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
Manufacturer: Agilent<br> <br />
Column number: 19091Z-433<br><br />
Stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds<br />
based on their differences in mass-to-charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that<br />
the separate substrates in the machine will be bombarded with electrons in a vaccuum. Because of this, they will receive a charge and are most likely <br />
to break into pieces. These flying bits and pieces of substrates, each with their own charge, will be prevented from crashing into the wall by the<br />
quadruple poles. One can adjust the changing charge of the poles and thereby choose the range of spectrum of the identifiable compounds with their <br />
different mass-to-charge ratios.<br />
<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules (which might cause redundant mass charge ratios), <br />
and how small the differences in molecule structure are (like isomers) that are difficult to separate.<br />
<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra. Unfortunately, the concentration of volatiles was probably too low in the extracted <br />
sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles.<br />
In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine<br />
as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature.<br />
Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
<br><br />
But also this did not work out, because we got blank spectra again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better<br />
than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not give up, though.<br />
<br><br />
<br><br />
<z2>Liquid injection with organic solvents</z2><br />
<br><br />
<br><br />
The first measurements with only volatiles gave no reliable results. Therefore, we decided to dissolve the rotten meat into organic solvents to ensure that<br />
more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot<br />
for more than a day at room temperature before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent<br />
for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent;<br />
we needed to study this thoroughly, and to cover all the polar and non-polar volatiles, we used different organic solvents.<br />
<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat. In order to prevent damage to the columns <br />
we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br />
<br><br />
<br> <br />
<z2>Results</z2><br />
<br><br />
<br><br />
Finally, this method produced interesting results from both the HP-1 and HP-5 column spectra. After the library search; only compounds <br />
with a quality over 80% were considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra<br />
of fresh meat and the solvent blank. Subtracting the solvent blank removes the background noise, while subtracting the fresh meat highlights those spectra <br />
up- and downregulated in rotten-meat volatiles. This approach identified the following compounds in rotten meat, <br />
with a quality that matches with approximately 80% to the reference library.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width=350 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" class="preview" width=600 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width=350 /><br />
<img src="https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" class="preview" width=600 /><br />
</a><br />
</li><br />
</ul><br />
<p><br />
<br><br />
We discussed our results with Prof. Dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 <br />
fatty acid, which is expected not to come out of the columns easily. This substrate is also found on both columns and with different solvents.<br />
<br><br />
<br><br />
He also noted that the overall reference quality was low because results with less than 80% quality are considered as unreliable. This is probably caused by compounds<br />
that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br />
<br><br />
<br> <br />
Some of the compounds found contain a fluoro group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded<br />
these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrates are also not likely to be correct. An explanation for this <br />
lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to <br />
known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer ‘blindly’ decides what the best fitting substrate spectra is. <br />
These fits can be incorrect, so we should take care when extracting conclusions from these results.<br />
<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other <br />
interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br />
<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra are <br />
compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform <br />
more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, not the major part. We decided to spend our time on other research <br />
although Prof. Minnaard suggested that we should do another microarray experiment using these compounds instead of the rotten meat.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxM"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxR"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<p class="caption"><br />
GC-MS spectra using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.<br />
<br><br />
<br><br />
</p><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxM"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<ul class="hoverboxR"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /><br />
</a><br />
</li><br />
</ul><br />
<p class="caption"><br />
GC-MS spectra using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Future plans</z2><br />
<br><br />
<br><br />
If time allows, we will try to set up an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. <br />
Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the<br />
growth of <i>B. subtilis</i> with our construct.<br />
<br><br />
<br><br />
<z2>Reference and acknowledgement</z2><br />
<br><br />
<br><br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry <br />
for her valuable help during the measurements and her explanations about the technique.<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/SensorTeam:Groningen/Sensor2012-10-26T18:03:51Z<p>Jparrish: </p>
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<z1>Sensor</z1><br />
</div><br />
</div><br />
<p><br />
We took a long journey in finding a sensor for spoiled meat. After a literature research, we decided to find a <br />
spoilage sensor experimentally. Read about our quest for "P<sub>BADmeat</sub>" below.<br />
<br><br />
<br><br />
<z2>A literature study about the alsT promoter</z2><br />
<br><br />
<br><br />
The promoter that was initially chosen for the food warden construct was the alsT promoter. This gene is one of several genes that is regulated by TnrA. <br />
TnrA is a regulator in <i>B. subtilis</i> that is triggered by the availability of nitrogen sources. It regulates a few genes and one of them that is <br />
repressed is alsT. TnrA is only active when the amount of nitrogen is low. Since alsT is repressed by TnrA, we suggested that it gets activated as soon<br />
as TnrA is depleted due to the availability of nitrogen (Kayumov <i>et al.,</i> 2008). There are a few components in rotting meat that contain nitrogen. <br />
One of these components is ammonia, a highly volatile and abundant compound. These volatiles would trigger the depletion of TnrA, removing it from the <br />
alsT promoter, activating the meat warden construct. <br />
<br><br />
<br><br />
We identified this promoter-regulator system only by performing a literature study. And it was given that there were only a few components in rotting meat<br />
that contain nitrogen. One of these components is ammonia, a highly volatile and abundant compound. These volatiles would trigger the depletion of TnrA, <br />
removing it from the alsT promoter, activating the pBAD meat promoters. However, we were not completely convinced that our whole project should depend <br />
on one promoter, so we asked our modeler to give us more insight into the regulator TnrA and its behavior when exposed to nitrogen sources. Unfortunately, <br />
the results on the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Modeling" target=_blank">modeling page</a> were disappointing…<br />
</p><br />
<br><br />
<z4>Reference</z4><br />
<p class=ref><br />
Kayumov, A. Heinrich, A. Sharipova, M. Iljinskaya, O. Forchhammer, K. Inactivation of the general transcription factor TnrA in Bacillus subtilis by proteolysis,<br />
Microbiology (2008), 154, 2348–2355.<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Finding P<sub>BADmeat</sub> by transcriptome analysis</z2><br />
<br><br />
<br><br />
With the literature research reaching a dead end and the weeks passing by, we had to come up with a different strategy for finding a sensor for rotting meat volatiles.<br />
<br><br />
<br><br />
We decided to see whether <i> Bacillus subtilis sp. 168</i> would react to spoiled meat itself. This had two major reasons: the first is that<br />
<i>Bacillus subtilis</i> naturally inhabits an environment (especially soil), where it is plausible that it can detect certain metabolites <br />
from other organisms. Furthermore, if we would find a promoter upregulated in <i>B. subtilis</i>, this would make the construct inside our chassis<br />
more reliable than when we would have to transfer a whole uptake system in the not-so-easily-tweaked <i>B. subtilis</i>.<br />
<br><br />
<br><br />
<z2>Microarray analysis</z2><br />
<br><br />
<br> <br />
We compare the difference in transcription level of all genes (the transcriptome) of <i>Bacillus subtilis </i> at a certain time point, <br />
at defined conditions. We compared the difference in transcription in the exponential growth phase of <i>B. subtilis</i> subjected to fresh and to rotten meat.<br />
<br><br />
<br><br />
From both test conditions, we harvested the mRNA of <i>Bacillus subtilis</i>. With reverse transcriptase, the mRNA was copied into cDNA. Half of the cDNA <br />
was labeled with a green dye, the other half with a red dye. The cDNA of both conditions was brought onto a microarray slide: one red, the other green. <br />
On this slides, small parts (probes) of DNA are put at a known position. The cDNA hybridized with the DNA probes. Because of the colored label, we could <br />
define which genes were transcribed in the green condition, and which genes were transcribed in the red condition. The ratio green:red is a measure for the <br />
up- or downregulation of the genes. On each slide there are three technical replicates to increase the significance of the data. Using a second slide we <br />
swapped the dyes bound to de cDNA of both conditions, in order to rule out any error in dye binding and verifying the acquired data. We also did the whole <br />
experiment in duplo to add significance to the data and avoiding artifacts. The entire workflow is shown below:<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/89/Groningen2012_ID2_0120925_mic_array_slide.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<z2>Experimental setup</z2><br />
<br><br />
<br><br />
How did we measure the transcriptome of <i>Bacillus subtilis</i> subjected to meat volatiles? We had to grow <i>Bacillus subtilis</i> in the presence of meat, <br />
without touching the meat or interfering with the growth of <i>Bacillus subtilis</i>. We tried a few setups (also fermentors). In the end we decided to use <br />
simple closed system, with a pot with rotten meat (rotten in fridge for 7 days), from which the air (filtered) goes through bacterial culture, stirred with a <br />
magnetic stirrer. The air is pumped through the system with a simple peristaltic pump. The whole system was running at 37 degrees Celsius, note: both the fresh<br />
and rotted meat is placed on ice, this prevented the rotting of the fresh meat during the experiment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_ID2_0120925_Micarraysetup_realpicture.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Experimental setup for meat testing. Volatiles of rotten or fresh meat were flushed through a culture of <i>Bacillus subtilis</i> for two hours. <br />
After this, RNA was harvested for further analysis. Check the system in real life below! <br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/LM3el_V-GNk"></iframe><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br />
<br><br />
<br><br />
<z2>Growth conditions</z2><br />
We inoculated <i>Bacillus subtilis</i> from plate and let it grow over night in Luria Broth, in a normal shake flask (37 degrees Celsius). <br />
We diluted the culture to OD(600) = 0.3 and let it grow in a normal shake flask (37 degrees Celsius) until the OD(600) was 0.8 (approximately 2 hours).<br />
At that point, the cultures was diluted to OD(600) = 0.1 and put it in our experimental setup. The setup was ran for two hours, until the OD(600) = 0.8.<br />
<br><br />
<br><br />
<z2>Analysis</z2><br />
We analyzed the slide images using ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, MD). The obtained data was processed and normalized (Lowess normalization) <br />
using the in-house software of the Molecular Genetics group MicroPrep (Van Hijum et. al, 2003). A statistical analysis was done using the webservice CyberT <br />
(Baldi et. al, 2001) A gene was considered differentially expressed when the Bayes p value was lower than 0.001 and the difference in expression (the fold)<br />
was >2 or <-2. The genes obtained using the CyberT analysis were ordered by locus tag and fold. This list was processed further using different analysis tools. <br />
With use of MolGen’s in-house software Genome2d (Baerends et. al, 2004), operons with expression difference were identified and figures showing these operons were made.<br />
<br><br />
<br><br />
<z2>Results</z2><br />
<br><br />
We found 19 upregulated operons. We had to rule many of them out, because they were somehow involved in stress-reactions and regulated by general transcription <br />
factors like SigmaB. This left us with five upregulated operons. Of this, we took the two highest upregulated operons, the <i>NarK-fnr</i> and <i>sboA-sboX-Alb</i> operon.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Groningen2012_ID_20120925_sboa_fnr_information.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<i>NarK-fnr</i> is regulated by the redox regulator Fnr. In <i>Bacillus subtilis</i>, Fnr induces the expression of the <i>narGHJI</i> operon as well as <i>NarK</i>, <br />
which are both highly upregulated in our bacteria exposed to meat. The <i>fnr</i> gene is known to be induced in an anaerobic environment in the presence of nitrate. <br />
Since the experimental setup we used in the control (fresh meat) as well as the target (spoiled meat) is a closed system, there is a shortage of oxygen in both situations. <br />
The reason that the operon is upregulated should therefore be a difference in nitrate or other, unknown reactions due to the rotten meat.<br />
<br><br />
<br><br />
Both <i>fnr</i> and <i>sboA-sboX-Alb</i> are regulated by the ResDE signal transduction system. It is thought that this system only activates anaerobically induced <br />
genes in the presence of nitrite. This might explain the difference between the target and control.<br />
<br><br />
<sboA> encodes the production of an antibiotic compound called subtilosin. It is produced by <i>Bacillus subtilis</i> at anaerobic conditions and at very high cell <br />
densities. Apart from regulation by ResDE, it can also be regulated by the Spo0A system.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Groningen2012_ID2_0120925_mntc_wapa_downregulation.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<z2>Expression details of downregulated operons <i>WapA-yxxG</i> and <i>mntC-mntB-mntA</i>.</z2><br />
<br><br />
<br><br />
There were also many downregulated operons. Most of these operons where general stress responses, but there were two interesting operons involved<br />
in high salt concentrations (<i>mntC-mntB-mntA</i> and salt stress: <i>WapA-yxxG</i>). The first is repressed at high Mn(II) concentrations by MntR,<br />
while it is normally activated by SigmaB and TnrA (see the AlsT section). The second is a two-component system which is repressed at high salt concentrations<br />
by DegU-P. WapA is a cellwall-associated protein which is known to be highly repressed in the presence of 0.7 M disodium succinate. It is thought that <br />
another unknown repressor controls the downregulation of wapA as well. These downregulated operons could be interesting for future implementation into<br />
a multi-colored system (see our <A HREF=https://2012.igem.org/Team:Groningen/in_development><FONT COLOR=#ff6700>development page</FONT></A>).<br />
<br><br />
<br><br />
<z2>Testing the promoters</z2><br />
<br><br />
<br><br />
We tested the promoters using our micro-array setup. You can find the test and the results on our <br />
<a class="inlink" href=https://2012.igem.org/Team:Groningen/Construct>construct page</a>.<br />
<br><br />
<br><br />
</p><br />
<z4>Reference</z4><br />
<p class=ref><br />
<ol class="ref"><br />
<li>Reents H., Münch R., Dammeyer T., Jahn D., Härtig E. (2005).The Fnr Regulon of <i>Bacillus subtilis</i>. Journal of Bacteriology, 188(3):1103-1112</li><br />
<br />
<li>Nakano M. M., Zheng G., Zuber P. (2000). Dual control of <i>sboa-alb</i> operon expression by the Spo0 and ResDE systems of signal transduction under anaerobic conditions in <i>Bacillus subtilis</i>. Journal of Bacteriology, 181(11): 3274-3277</li><br />
<br />
<li>Dartois V. Débarbouillé M., Kunst F., Rapoport G. (1998). Characterization of a novel member of the DegS-DegU regulon affected by salt stress in <i>Bacillus subtilis</i>. Journal of Bacteriology, 180(7): 1855-1861</li><br />
<br />
<li>Serizawa M., Kodama K, Yamamoto Hl, Kobayashi K., Oqasawara N., Sekiquchi J. (2005) Functional analysis of the YvrGHb two-component system of <i>Bacillus subtilis</i>: identification of the regulated genes by DNA microarray and northern blot analyses. Bioscience, biotechnology and biochemistry, 69(11): 2155-2169</li><br />
<br />
<li>Baldi , Lond, A. D. (2001): "A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes", Bioinformatics 17(6):509-519.</li><br />
<br />
<li>Baerends R, Smits W, De Jong A, Hamoen L, Kok J, Kuipers O (2004). "Genome2D: a visualization tool for the rapid analysis of bacterial transcriptome data." Genome Biol, 5(5):R37.</li><br />
</ol><br />
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</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/SensorTeam:Groningen/Sensor2012-10-26T18:02:24Z<p>Jparrish: </p>
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<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Sensor</z1><br />
</div><br />
</div><br />
<p><br />
We took a long journey in finding a sensor for spoiled meat. After a literature research, we decided to find a <br />
spoilage sensor experimentally. Read about our quest for "P<sub>BADmeat</sub>" below.<br />
<br><br />
<br><br />
<z2>A literature study about the alsT promoter</z2><br />
<br><br />
<br><br />
The promoter that was initially chosen for the food warden construct was the alsT promoter. This gene is one of several genes that is regulated by TnrA. <br />
TnrA is a regulator in <i>B. subtilis</i> that is triggered by the availability of nitrogen sources. It regulates a few genes and one of them that is <br />
repressed is alsT. TnrA is only active when the amount of nitrogen is low. Since alsT is repressed by TnrA, we suggested that it gets activated as soon<br />
as TnrA is depleted due to the availability of nitrogen (Kayumov <i>et al.,</i> 2008). There are a few components in rotting meat that contain nitrogen. <br />
One of these components is ammonia, a highly volatile and abundant compound. These volatiles would trigger the depletion of TnrA, removing it from the <br />
alsT promoter, activating the meat warden construct. <br />
<br><br />
<br><br />
We identified this promoter-regulator system only by performing a literature study. And it was given that there were only a few components in rotting meat<br />
that contain nitrogen. One of these components is ammonia, a highly volatile and abundant compound. These volatiles would trigger the depletion of TnrA, <br />
removing it from the alsT promoter, activating the pBAD meat promoters. However, we were not completely convinced that our whole project should depend <br />
on one promoter, so we asked our modeler to give us more insight into the regulator TnrA and its behavior when exposed to nitrogen sources. Unfortunately, <br />
the results on the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Modeling" target=_blank">modeling page</a> were disappointing…<br />
</p><br />
<br><br />
<z4>Reference</z4><br />
<p class=ref><br />
Kayumov, A. Heinrich, A. Sharipova, M. Iljinskaya, O. Forchhammer, K. Inactivation of the general transcription factor TnrA in Bacillus subtilis by proteolysis,<br />
Microbiology (2008), 154, 2348–2355.<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Finding P<sub>BADmeat</sub> by transcriptome analysis</z2><br />
<br><br />
<br><br />
With the literature research reaching a dead end and the weeks passing by, we had to come up with a different strategy for finding a sensor for rotting meat volatiles.<br />
<br><br />
<br><br />
We decided to see whether <i> Bacillus subtilis sp. 168</i> would react to spoiled meat itself. This had two major reasons: the first is that<br />
<i>Bacillus subtilis</i> naturally inhabits an environment (especially soil), where it is plausible that it can detect certain metabolites <br />
from other organisms. Furthermore, if we would find a promoter upregulated in <i>B. subtilis</i>, this would make the construct inside our chassis<br />
more reliable than when we would have to transfer a whole uptake system in the not-so-easily-tweaked <i>B. subtilis</i>.<br />
<br><br />
<br><br />
<z2>Microarray analysis</z2><br />
<br><br />
<br> <br />
We compare the difference in transcription level of all genes (the transcriptome) of <i>Bacillus subtilis </i> at a certain time point, <br />
at defined conditions. We compared the difference in transcription in the exponential growth phase of <i>B. subtilis</i> subjected to fresh and to rotten meat.<br />
<br><br />
<br><br />
From both test conditions, we harvested the mRNA of <i>Bacillus subtilis</i>. With reverse transcriptase, the mRNA was copied into cDNA. Half of the cDNA <br />
was labeled with a green dye, the other half with a red dye. The cDNA of both conditions was brought onto a microarray slide: one red, the other green. <br />
On this slides, small parts (probes) of DNA are put at a known position. The cDNA hybridized with the DNA probes. Because of the colored label, we could <br />
define which genes were transcribed in the green condition, and which genes were transcribed in the red condition. The ratio green:red is a measure for the <br />
up- or downregulation of the genes. On each slide there are three technical replicates to increase the significance of the data. Using a second slide we <br />
swapped the dyes bound to de cDNA of both conditions, in order to rule out any error in dye binding and verifying the acquired data. We also did the whole <br />
experiment in duplo to add significance to the data and avoiding artifacts. The entire workflow is shown below:<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/89/Groningen2012_ID2_0120925_mic_array_slide.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<z2>Experimental setup</z2><br />
<br><br />
<br><br />
How did we measure the transcriptome of <i>Bacillus subtilis</i> subjected to meat volatiles? We had to grow <i>Bacillus subtilis</i> in the presence of meat, <br />
without touching the meat or interfering with the growth of <i>Bacillus subtilis</i>. We tried a few setups (also fermentors). In the end we decided to use <br />
simple closed system, with a pot with rotten meat (rotten in fridge for 7 days), from which the air (filtered) goes through bacterial culture, stirred with a <br />
magnetic stirrer. The air is pumped through the system with a simple peristaltic pump. The whole system was running at 37 degrees Celsius, note: both the fresh<br />
and rotted meat is placed on ice, this prevented the rotting of the fresh meat during the experiment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_ID2_0120925_Micarraysetup_realpicture.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Experimental setup for meat testing. Volatiles of rotten or fresh meat were flushed through a culture of <i>Bacillus subtilis</i> for two hours. <br />
After this, RNA was harvested for further analysis.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/LM3el_V-GNk"></iframe><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Movie: Check the system in real life! <br />
<br><br />
<br><br />
<z2>Growth conditions</z2><br />
We inoculated <i>Bacillus subtilis</i> from plate and let it grow over night in Luria Broth, in a normal shake flask (37 degrees Celsius). <br />
We diluted the culture to OD(600) = 0.3 and let it grow in a normal shake flask (37 degrees Celsius) until the OD(600) was 0.8 (approximately 2 hours).<br />
At that point, the cultures was diluted to OD(600) = 0.1 and put it in our experimental setup. The setup was ran for two hours, until the OD(600) = 0.8.<br />
<br><br />
<br><br />
<z2>Analysis</z2><br />
We analyzed the slide images using ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, MD). The obtained data was processed and normalized (Lowess normalization) <br />
using the in-house software of the Molecular Genetics group MicroPrep (Van Hijum et. al, 2003). A statistical analysis was done using the webservice CyberT <br />
(Baldi et. al, 2001) A gene was considered differentially expressed when the Bayes p value was lower than 0.001 and the difference in expression (the fold)<br />
was >2 or <-2. The genes obtained using the CyberT analysis were ordered by locus tag and fold. This list was processed further using different analysis tools. <br />
With use of MolGen’s in-house software Genome2d (Baerends et. al, 2004), operons with expression difference were identified and figures showing these operons were made.<br />
<br><br />
<br><br />
<z2>Results</z2><br />
<br><br />
We found 19 upregulated operons. We had to rule many of them out, because they were somehow involved in stress-reactions and regulated by general transcription <br />
factors like SigmaB. This left us with five upregulated operons. Of this, we took the two highest upregulated operons, the <i>NarK-fnr</i> and <i>sboA-sboX-Alb</i> operon.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
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<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Groningen2012_ID_20120925_sboa_fnr_information.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<i>NarK-fnr</i> is regulated by the redox regulator Fnr. In <i>Bacillus subtilis</i>, Fnr induces the expression of the <i>narGHJI</i> operon as well as <i>NarK</i>, <br />
which are both highly upregulated in our bacteria exposed to meat. The <i>fnr</i> gene is known to be induced in an anaerobic environment in the presence of nitrate. <br />
Since the experimental setup we used in the control (fresh meat) as well as the target (spoiled meat) is a closed system, there is a shortage of oxygen in both situations. <br />
The reason that the operon is upregulated should therefore be a difference in nitrate or other, unknown reactions due to the rotten meat.<br />
<br><br />
<br><br />
Both <i>fnr</i> and <i>sboA-sboX-Alb</i> are regulated by the ResDE signal transduction system. It is thought that this system only activates anaerobically induced <br />
genes in the presence of nitrite. This might explain the difference between the target and control.<br />
<br><br />
<sboA> encodes the production of an antibiotic compound called subtilosin. It is produced by <i>Bacillus subtilis</i> at anaerobic conditions and at very high cell <br />
densities. Apart from regulation by ResDE, it can also be regulated by the Spo0A system.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Groningen2012_ID2_0120925_mntc_wapa_downregulation.png" width="700"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
<z2>Expression details of downregulated operons <i>WapA-yxxG</i> and <i>mntC-mntB-mntA</i>.</z2><br />
<br><br />
<br><br />
There were also many downregulated operons. Most of these operons where general stress responses, but there were two interesting operons involved<br />
in high salt concentrations (<i>mntC-mntB-mntA</i> and salt stress: <i>WapA-yxxG</i>). The first is repressed at high Mn(II) concentrations by MntR,<br />
while it is normally activated by SigmaB and TnrA (see the AlsT section). The second is a two-component system which is repressed at high salt concentrations<br />
by DegU-P. WapA is a cellwall-associated protein which is known to be highly repressed in the presence of 0.7 M disodium succinate. It is thought that <br />
another unknown repressor controls the downregulation of wapA as well. These downregulated operons could be interesting for future implementation into<br />
a multi-colored system (see our <A HREF=https://2012.igem.org/Team:Groningen/in_development><FONT COLOR=#ff6700>development page</FONT></A>).<br />
<br><br />
<br><br />
<z2>Testing the promoters</z2><br />
<br><br />
<br><br />
We tested the promoters using our micro-array setup. You can find the test and the results on our <br />
<a class="inlink" href=https://2012.igem.org/Team:Groningen/Construct>construct page</a>.<br />
<br><br />
<br><br />
</p><br />
<z4>Reference</z4><br />
<p class=ref><br />
<ol class="ref"><br />
<li>Reents H., Münch R., Dammeyer T., Jahn D., Härtig E. (2005).The Fnr Regulon of <i>Bacillus subtilis</i>. Journal of Bacteriology, 188(3):1103-1112</li><br />
<br />
<li>Nakano M. M., Zheng G., Zuber P. (2000). Dual control of <i>sboa-alb</i> operon expression by the Spo0 and ResDE systems of signal transduction under anaerobic conditions in <i>Bacillus subtilis</i>. Journal of Bacteriology, 181(11): 3274-3277</li><br />
<br />
<li>Dartois V. Débarbouillé M., Kunst F., Rapoport G. (1998). Characterization of a novel member of the DegS-DegU regulon affected by salt stress in <i>Bacillus subtilis</i>. Journal of Bacteriology, 180(7): 1855-1861</li><br />
<br />
<li>Serizawa M., Kodama K, Yamamoto Hl, Kobayashi K., Oqasawara N., Sekiquchi J. (2005) Functional analysis of the YvrGHb two-component system of <i>Bacillus subtilis</i>: identification of the regulated genes by DNA microarray and northern blot analyses. Bioscience, biotechnology and biochemistry, 69(11): 2155-2169</li><br />
<br />
<li>Baldi , Lond, A. D. (2001): "A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes", Bioinformatics 17(6):509-519.</li><br />
<br />
<li>Baerends R, Smits W, De Jong A, Hamoen L, Kok J, Kuipers O (2004). "Genome2D: a visualization tool for the rapid analysis of bacterial transcriptome data." Genome Biol, 5(5):R37.</li><br />
</ol><br />
</p><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:39:06Z<p>Jparrish: </p>
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<z1>Pigments</z1><br />
</div><br />
</div><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:36:47Z<p>Jparrish: </p>
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<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
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<div style="position:absolute; right: 0px; bottom: 760px;"><br />
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</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:36:08Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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</head><br />
<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Construct"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:33:56Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br />
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<head><br />
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</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Construct"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:32:32Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<br />
<br />
<br />
<html><br />
<head><br />
<style><br />
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</style><br />
</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Construct"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:31:26Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<br />
<html><br />
<head><br />
<style><br />
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</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
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<div style="position:absolute; right: 0px; bottom: 760px;"><br />
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</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
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</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:30:29Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
</p><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Construct"><br />
<div style="position:absolute; right: 10px; bottom: 560px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 20px; bottom: 500px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:28:37Z<p>Jparrish: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<head><br />
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</head><br />
<body><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
<br><br />
<br><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/pigmentproductionTeam:Groningen/pigmentproduction2012-10-26T17:27:49Z<p>Jparrish: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Pigments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<p><br />
<br><br />
<br><br />
We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common<br />
reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system as a consumer product. <br />
Therefore, it is unreasonable to expect everyone to have a fluorescence microscope available at home. Pigments, however, <br />
are recognizable by the naked eye and come in many color varieties, but a main challenge we had to accept was that <br />
these pigments had yet to be expressed <i>Bacillus subtilis </i>. We decided to take the risk, and look the great results!<br />
<br><br />
<br><br />
We identified many pigments in the Registry and choose three BioBricks that we could express independently <br />
so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:<br />
<br><br />
<br><br />
</p><br />
<p><br />
<z2>Lycopene (BBa_K274100)</z2><br />
<br><br />
<br><br />
Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter genes in<br />
our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.<br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K274100">BBa_K274100</a>.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by <font color=#ff6700>LMU Munich 2012</font>), to allow color<br />
expression in <i>B. subtilis</i>. We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/Groningen2012_AP20120924_FnrLycopene.png" width="500" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Groningen2012_AP20120924_BsPromoterslycopene.png" width="500"><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>B. subtilis</i> 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as <br />
highly expressed as in <i>E. coli</i>. This phenotype was due to the weak promoter of <i>B. subtilis</i> that was used in this <br />
construct and the RBS for <i>E. coli</i> that was utilized for this device construct.<br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of <br />
the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. <br />
<a class="inlink" href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>. AmilCP has a sequence similar to the other <br />
coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>. <br />
We utilized a strong RBS BBa_B0034 for pigment expression in <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding gene was strongly<br />
expressed in <i>E. coli</i> due to the leakiness of the promoters. However, the expression in <i>B. subtilis</i> was not as <br />
leaky as in <i>E. coli</i>. This phenotype in <i>B. subtilis</i> was due to the promoter strength and the RBS that was used in <br />
this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right" width="400px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Groningen2012_AP20120924_EcoliSboAamilCP.jpg" width="100" ><br />
</td><br />
<td align="left"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/Groningen2012_AP20120924_BsPromotersamilCP_small.png" width="400" ><br />
</td><br />
</tr><br />
</table><br />
<p> <br />
<i>E. coli</i> DH5a containing sboA promoter + amilCP (left). Blue/purple colour was highly <br />
expressed due to the leakiness of the promoter and a strong RBS for <i>E. coli</i>. However, the expression <br />
of this part in <i>B. subtilis</i> was more subtle. <i>B. subtilis</i> containing sboA promoter + amilCP <br />
(right) displayed faint blue colour on its colony. <br />
<br><br />
<br> <br />
<z2>AmilCP (BBa_K592009)</z2><br />
<br><br />
<br><br />
AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used <br />
as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala <br />
Sweden 2011 page. <a class="inlink" href="http://partsregistry.org/Part:BBa_K592010">BBa_K592010</a>. <br />
This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.<br />
<br> <br />
<br><br />
Our team has coupled this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 <br />
(plasmid backbone for <i>B. subtilis</i>, engineered by team Groningen 2012), to allow color expression in <i>B. subtilis</i>.<br />
We utilized a strong RBS BBa_B0034 for pigment expression in both <i>E. coli</i> and <i>B. subtilis</i>. This reporter coding <br />
gene was strongly expressed in <i>E. coli</i> due to the leakiness of the promoters, while the expression in B. subtilis without <br />
induction was considerably low. This low expression in <i>B. subtilis</i> was due to the promoter strength and the RBS that was <br />
used in this construct. The promoters are considered weak, therefore visible expression requires induction by volatiles produced<br />
by the rotten meat.<br />
<br><br />
<br><br />
The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. <br />
We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, when<br />
our device that was exposed to the volatiles produced by the fresh meat it produced a small amount of yellow color. The <br />
comparison between <i>B. subtilis</i> containing sboA + amilGFP to <i>B. subtilis</i> 168 wildtype under the presence of rotten<br />
meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. <br />
The yellow color produced by our device was strong enough to be observed by the human naked eye.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="100" ><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<i>E. coli</i> DH5a containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the<br />
promoter and a strong RBS for <i>E. coli</i>.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="right"><br />
<a href="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_AP20120924_sboAamilGFPsetup_small.png" width="350"><br />
</a><br />
</td><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Groningen2012_AP20120924_sboAamilGFPsetupcell_small.png" width="350"><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Our setup for the volatile detection experiment using the sboA+amilGFP device (left). We compared the<br />
pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat.<br />
When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the<br />
amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun <br />
down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles<br />
exhibited strong yellow color while the wildtype strain and the device that was not exposed to the volatiles were white (right).<br />
<br><br />
<br><br />
</p><br />
</body><br />
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<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
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</html></div>Jparrishhttp://2012.igem.org/Team:Groningen/acknowledgementsTeam:Groningen/acknowledgements2012-10-26T16:30:01Z<p>Jparrish: </p>
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<z1>Acknowledgments</z1><br />
</div><br />
</div><br />
<br><br />
<br><br />
<table class="margintable"><br />
<tr><br />
<td><br />
<table class="info"><br />
<tr colspan="2"><br />
<td class="textcell"><br />
<z5>We want to thank everybody who helped us during the project, especially:</z5><br />
</td><br />
</tr><br />
<tr><br />
<td class="textcell"><br />
<z6>Our supervisors and advisors</z6><br />
<br><br />
<br><br />
<z6>Leen van Wijngaarden</z6>, <z6>Jan Kiel</z6>, and <z6>Christa Holtkamp</z6> for making it possible that we could paint Bio art<br />
<br><br />
<br><br />
<z6>Ger Telkamp</z6> for providing us with lab equipment<br />
<br><br />
<br><br />
<z6>Sjoerd Murris</z6>, Science Linx - Summerschool<br />
<br><br />
<br><br />
<z6>Monique Smith</z6> and <z6>Prof. A.J. Minnaard</z6> for their help with the Gas Chromatography-Mass Spectrometry experiments and the data analysis<br />
<br><br />
<br><br />
We were advised by <z6>Dr. J. Lolkema</z6> and <z6>Prof. dr. ir. J.D van Elsas</z6> concerning our safety page<br />
<br><br />
<br><br />
<z6>Gert-Jan Euverink</z6> for sharing ideas on the sticker material<br />
<br><br />
<br><br />
<z6>Jan-Willem Veening</z6>, <z6>Jeroen Siebring</z6>, <z6>Tonia</z6>, <z6>Katrin</z6> because of their advice how to use the fluorescence microscope<br />
<br><br />
<br><br />
<z6>Anne de Jong</z6>, who advised us on the microarray experiments<br />
<br><br />
<br><br />
<z6>Anne Hesseling</z6>, for her help with finances<br />
<br><br />
<br><br />
<z6>Martijn Herber</z6>, <z6>Wout Overkamp</z6>, and many others of Molecular Genetics<br />
<br><br />
<br><br />
<z6>The Molecular Microbiology Group of the RUG</z6>, for letting us borrow the peristaltic pump and providing us with fresh impact on our project presentation<br />
<br><br />
</td><br />
</tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
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{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/AttributionsTeam:Groningen/Attributions2012-10-26T16:24:31Z<p>Jparrish: </p>
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<z1>Attributions</z1><br />
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<br><br />
<br><br />
<p><br />
All the work for this project was performed by the iGEM Groningen 2012 team members, the lab work started from May 2012 onwards.<br />
<br><br />
<br><br />
The original <i>Bacillus subtilis</i> integration vector pSac-Cm from which we derived our backbone biobrick originates from the article by Middleton et al. [1] and was provided <br />
by Dr. Jan-Willem Veening from the Molecular Genetics group of the University of Groningen, as was the GFP optimized for<br />
<i>Bacillus subtilis</i> for our testing constructs, the <i>Escherichia coli dh5a</i> strain and the <i>Bacillus subtilis sp. 168</i> strain we used in the lab.<br />
<br><br />
<br><br />
The idea of the Food Warden was developed by the team before the labwork started. All experiments, the modeling and the<br />
sticker design were planned and carried out by the team. Directions were given by our advisors, but they <br />
only had a consultative function.<br />
<br><br />
<br><br />
We were informed by Marius Uebel about GMP/SOP and data management with the use of acronyms. The team also took care of <br />
the sponsoring and all the other organizational issues. We made our own logos, drawings and wiki/presentation/poster style. <br />
The picture gallery on the home page and in other sections of our wiki has been made using the hoverbox image gallery code by<br />
<a class="inlink" href="http://sonspring.com/journal/hoverbox-image-gallery">Nathan Smith</a> as a basis.<br />
<br><br />
<br> Last but not least, we wanted to add an extra feature to the wiki, by providing everyone with an exact copy of the iGEM server for use <br />
on a local hard disk. Each team can immediately see the versions of all the available libraries now. Click <br />
<a class="inlink" href="http://igem.molgenrug.nl/iGEM2012/IGEM_Porable_wiki_2012_Groningen.rar">here</a>.<br />
</p><br />
<p class="ref"><br />
[1] Middleton et al. [1], R., Hofmeister, A. New shuttle vectors for ectopic insertion of genes into Bacillus subtilis. Plasmid Volume 51, Issue 3, May 2004, Pages 238–245<br> <br />
<br><br />
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{{Template:SponsorsGroningen2012}}</div>Jparrishhttp://2012.igem.org/Team:Groningen/acknowledgementsTeam:Groningen/acknowledgements2012-10-26T16:21:44Z<p>Jparrish: </p>
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<z1>Acknowledgments</z1><br />
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<z5>We want to thank everybody who helped us during the project, especially:</z5><br />
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<z6>Our supervisors and advisors</z6><br />
<br><br />
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<z6>Leen van Wijngaarden</z6>, <z6>Jan Kiel</z6>, and <z6>Christa Holtkamp</z6> for making it possible that we could paint Bio art<br />
<br><br />
<br><br />
<z6>Ger Telkamp</z6> for providing us with lab equipment<br />
<br><br />
<br><br />
<z6>Sjoerd Murris</z6>, Science Linx - Summerschool<br />
<br><br />
<br><br />
<z6>Monique Smith</z6> and <z6>Prof. A.J. Minnaard</z6> for their help with the Gas Chromatography-Mass Spectrometry experiments and the data analysis<br />
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We were advised by <z6>Dr. J. Lolkema</z6> and <z6>Prof. dr. ir. J.D van Elsas</z6> concerning our safety page<br />
<br><br />
<br><br />
<z6>Gert-Jan Euverink</z6> for sharing ideas on the sticker material<br />
<br><br />
<br><br />
<z6>Jan-Willem Veening</z6>, <z6>Jeroen Siebring</z6>, <z6>Tonia</z6>, <z6>Katrin</z6> because of their advice how to use the fluorescence microscope<br />
<br><br />
<br><br />
<z6>Anne de Jong</z6>, who advised us on the microarray experiments<br />
<br><br />
<br><br />
<z6>Anne Hesseling</z6>, for her help with finances<br />
<br><br />
<br><br />
<z6>Martijn Herber</z6>, <z6>Wout Overkamp</z6>, and many others of Molecular Genetics<br />
<br><br />
<br><br />
<z6>The Molecular Microbiology Group of the RUG</z6>, for letting us borrow the peristaltic pump and providing us with fresh impact on our project presentation<br />
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