Team:Groningen/Project

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== '''Overall project''' ==
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</head>
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More detailed abstract to come once the theory has been proven experimentally. For now, the homepage provides a broad overview.
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<body>
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<br>
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<div class="cte">
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<div class="ctd">
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<z1>Abstract</z1>
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</div>
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</div>
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<table class="centertable">
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<tr>
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<td class="margincell" align="right">
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<div class="bigcog" id="bigcogtopleft">
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px">
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</div>
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</td>
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<td colspan="4">
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<p class="nomargin">
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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.
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<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>
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of <i>Bacillus subtilis</i> to detect and report volatiles in spoiling meat. The introduced <z7>genetic construct</z7> uses a promoter to trigger
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a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of
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<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> and will
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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
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<z7>capsule</z7>, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows
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<z7>germination and growth</z7>, thereby activating the <z7>spoiling-meat sensor</z7>.
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</p>
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</td>
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<td class="margincell" align="left">
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<div class="bigcog" id="bigcogtopright">
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px">
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</div>
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</td>
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</tr>
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<tr>
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<td class="margincell">
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<div class="cogoverlay" id="cogoverlaybottomleft">
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<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100">
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</div>
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</td>
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<td align="left" >
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<div class="bigcog" id="bigcogbottomleft">
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px">
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</div>
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</td>
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<td align="left" >
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<div class="cogoverlay" id="cogoverlaytopleft">
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<img src="https://static.igem.org/mediawiki/2012/1/15/Groningen2012_RR20120909_construct.png"  width="100px" height="100px">
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</div>
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</td>
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<td align="right" >
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<div class="cogoverlay" id="cogoverlaytopright">
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<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width="100px" height="100px">
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</div>
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</td>
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<td align="right">
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<div class="bigcog" id="bigcogbottomright">
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150px" height="150px">
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</div>
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</td>
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<td class="margincell">
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<div class="cogoverlay" id="cogoverlaybottomright">
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<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100px" height="100px">
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</div>
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</td>
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</tr>
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</table>
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<br>
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<br>
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<div class="cte3">
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<div class="ctd3">
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<z2 >Completed after European Regionals</z2><br><br>
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</div>
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</div>
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<p class="marginChecklist">
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<br>
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<br>
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<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>
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<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>
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<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>
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<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>
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<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>
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<br>
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</p>
-
== Results ==
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<a name="MainAcc"></a>
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<div class="cte2">
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<div class="ctd2">
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<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br><br>
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</div>
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</div>
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<br>
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<br>
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<p align=center style="color: white; font-size: 10pt;">
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<i>Hover your mouse over the pictures to see more!</i>
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</p>
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<br>
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<table class="accompli">
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=== Video/animation and diagram of the critter in action ===
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<tr>
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<td class="accPic">
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<ul class="hoverbox">
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<li>
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<a href="#">
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<img src="https://static.igem.org/mediawiki/2012/c/cb/Groningen2012_RR1capsule_break.png" width=200 />
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<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_ADStickerBig.png" class="preview" width=400 />
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</a>
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</li>
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</ul>
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</td>
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<td class="accPic">
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<ul class="hoverbox">
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<li>
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<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>
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</li>
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</ul>
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</td>
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</tr>
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<tr>
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<td class="accCap">
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<p class="small">
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<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>.
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</p>
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</td>
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<td class="accCap">
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<p class="small">
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<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>
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</p>
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</td>
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</tr>
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<tr>
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<td class="accPic">
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<ul class="hoverbox">
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<li>
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<a href="#">
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<img src="https://static.igem.org/mediawiki/2012/2/29/Groningen2012_RR20120909_badmeat.png" width=200 />
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<img src="https://static.igem.org/mediawiki/2012/e/ec/Groningen2012_ADSensorBig.png" class="preview" width=400 />
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</a>
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</li>
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</ul>
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</td>
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<td class="accPic">
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<ul class="hoverbox">
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<li>
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<a href="#">
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<img src="https://static.igem.org/mediawiki/2012/2/24/Groningen2012_RRADPsac_transp.png" width=150 />
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<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_AdBackboneBig.png" class="preview" width=400 />
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</a>
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</li>
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</ul>
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</td>
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</tr>
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<tr>
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<td class="accCap">
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<p class="small">
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<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>
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</p>
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</td>
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<td class="accCap">
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<p class="small">
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<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.
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</p>
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</td>
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</tr>
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<tr>
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<td class="accPic">
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<ul class="hoverbox">
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<li>
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<a href="#">
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<img src="https://static.igem.org/mediawiki/2012/f/f1/Groningen2012_RRADPigments_transp.png" width=150 />
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<img src="https://static.igem.org/mediawiki/2012/3/35/Groningen2012_ADPigmentsBig.png"  class="preview" width=400 />
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</a>
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</li>
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</ul>
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</td>
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<td class="accPic">
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<ul class="hoverbox" style="margin-left: 30px">
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<li>
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<a href="#">
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<img src="https://static.igem.org/mediawiki/2012/9/93/Groningen2012_ADConstruct_yellow.png" width=220 />
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<img src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_ADConstructBig.png" class="preview" width=400 />
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</a>
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</li>
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</ul>
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</td>
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</tr>
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<tr>
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<td class="accCap">
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<p class="small">
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<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>.
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<br>
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These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.
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<br><br><br>
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</p>
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</td>
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<td class="accCap">
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<p class="small">
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<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.
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<br><br><br>
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</p>
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</td>
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</tr>
 +
</table>
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<br>
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<br>
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<br>
 +
</body>
 +
</html>
-
=== Model Analysis ===
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{{Template:SponsorsGroningen2012}}
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=== Experimental Analysis ===
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-
 
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== Project Details==
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-
 
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=== Theory ===
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'''Functionality'''
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-
<br>
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The monitor would be realized as a self-contained unit containing the sensing and reporting mechanisms.
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The sensing mechanism should allow monitoring of multiple foodstuffs at temperatures down to 4°C while having a response rate between 2 to 5 hours. The chassis must be able to survive long periods of inactivity.
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The reporter would utilize a color-coded system of identification, i.e. green is edible, red is not. Ideally, this should happen in a matter of minutes, but a combined detection/reporting response within 2 to 5 hours is sufficient.
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<br>
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<br>
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'''Sensing Mechanism'''
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<br>
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'''''Chassis'''''
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<br>
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The main factors in choosing a chassis are a prolonged dormant phase, ease of use, and the ability to survive in a cold environment. For this reason bacillus subtilis was selected. While it is not psychrotrophic (i.e. able to grow at <7oC), it has a dormant phase and is easy to use. It also allows for some novelty of using eukaryotic receptors in a prokaryotic chassis. Given a longer timeframe, the project methodology may be applied to detoxified strain of b. cereus, which is a psychrotrophic bacterium.
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<br>
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'''''Targets'''''
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{|
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| Amines || Trace amine-associated receptor 5 (TAAR5) - a eukaryotic G-coupled protein receptor
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|-
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| pH || Diffusion through the outer membrane
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|-
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| Ammonium || 1. NrgA ion channel at low concentrations 2. Diffusion through the outer membrane at high concentrations 
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|}
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<br>
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'''Reporters'''
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<br>
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'''''Indication methods'''''
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<br>
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There are a number of possibilities for changing the color of a bacterium, but the most suitable is the carotenoid pathway, either on its own or augmented with the tuning system developed for e.chromi by the 2009 Imperial College of London iGEM team. The carotenoid pathway provides a latched gradient from red to orange to yellow through the use of pigment.
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<br>
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<br>
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'''Enhancements'''
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<br>
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'''''Reaction time'''''
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# Amplify the production of the precursor and using an engineered enzymatic switch.
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# Adapt the fast-response mechanisms of the London yoghurt to e.chromi.
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'''''Thresholds and Sensitivity'''''
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<br>
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Histamine receptors need to calibrated to the total volume of meat (and volume of bacteria) within the package as histamine is always present.
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<br>
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A comprehensive set of experiments is necessary to determine the relationship between histamine concentration and spoilage rate. Once the relationship is known then the sensor mechanism can be tuned (if using e.chromi)
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Color progression
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<br>
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A switch is required to halt the red-to-orange and orange-to-yellow reactions The color change will use the color of the containment unit as a starting point (green). The indicator only need cover the base color as the indicator pigment is opaque.
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<br>
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<br>
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'''Challenges'''
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# Eukaryotic receptor in prokaryotic cell.
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# Tuning of the signaling and carotenoid pathways and the receptors threshold to levels appropriate for the quantity and geometry of the meat in the package.
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# Coupling the receptors to the carotenoid pathway.
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# Modification of the carotenoid pathway to disable the red-to-orange and orange-to-yellow transitions.
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<br>
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'''Limitations due to time constraints'''
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<br>
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# Food packages cannot be packed with a nitrogen atmosphere (no vacuum-packing). B. subtilis is aerobic.
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# Principles will be tested at temperatures above 7°C as working with B. cereus is too time consuming.
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==== Modeling ====
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===== Objectives =====
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===== Implementation =====
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===== Models =====
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===== Simulations =====
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===== Data =====
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=== Lab work ===
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==== Chassis and Vectors ====
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==== Functional Modules ====
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==== Protocols ====
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== Standard Operating Protocols ==
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In an effort to apply business concepts to the iGEM project we have agreed to conduct the project according to the following protocols. The specifics of these sections (such as the actual protocols or equipment listing) are contained within the SOP binder in the laboratory. This binder will be digitized for next year’s iGEM teams.
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=== General ===
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# How to set up an experiment.
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#* Fully describe the experiment in a document before it is scheduled to be performed (document is described in point 2).
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#* After the experiment is documented, it should be reviewed by at least one other iGEM member.
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#* Upon completion of the experiment there should be a short discussion/interpretation of the results and a short outlook for subsequent experiments.
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# Experiment Template
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#* Insert outline of the template here
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#Acronyms.
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#* Each iGEM member is assigned an acronym: e.g. Marius Uebel will be MU.
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# Data management.
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#* Each filename should contain the original creator, date, and type of file. E.g. MU_20120412_igem12_sop_proposal.doc
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# Ordering of material.
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#* A single person should be responsible for ordering, this prevents multiple orders of an item and a more controlled inventory.
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<br>
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=== Lab Equipment and Materials ===
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# Equipment.
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#* Every piece of lab equipment should be accompanied by the manufacturer’s manual and a short how-to manual.
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#* Overview and location of all our equipment should be written down in this part
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# Protocols
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#* All lab protocols should be consolidated into a central location in the lab in print form.
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<br>
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=== Methodology ===
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# Culture
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#* Contains general information and cultivation requirements of the chassis
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# Assay
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#* Short overview of any assay kits changes undertaken to suit the actual experiment.
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#* All information on original assays
+

Latest revision as of 02:00, 27 October 2012





Abstract

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. iGEM Groningen 2012 seeks to provide an alternative method of assessing edibility: the Food Warden. It uses an engineered strain of Bacillus subtilis to detect and report volatiles in spoiling meat. The introduced genetic construct uses a promoter to trigger a pigment coding gene. This promoter, identified by microarray analysis, is significantly upregulated in the presence of volatiles from spoiling meat. The activity of the promoter regulates the expression of the pigment reporter and will 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 capsule, the second containing a calibrated amount of nutrients. Breaking the barrier between the two compartments allows germination and growth, thereby activating the spoiling-meat sensor.



Completed after European Regionals



Tested a construct with downregulated promoter WapA
Performed enhanced construct characterization
Characterized the influence of oxygen on germination and growth within the sticker
Expanded the scope of the market survey
Collaborated with Cambridge 2012

Our main accomplishments



Hover your mouse over the pictures to see more!



1. We designed and tested the "Sticker": 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 Bacillus subtilis.

2. We explored the definition of spoiled meat and ways to check meat spoilage. We identified various compounds present in spoiled meat.

3. We identified spoiled meat sensors by transcriptome analysis.

4. Backbone pSac-Cm: easy cloning in
B. subtilis, the BioBrick way, for the first time! It's easy to check, BioBrick compatible, E. coli compatible, stabily inserted into the B. subtilis chromosome.



5. We made AmilCP and AmilGFP suitable for expression in Bacillus subtilis.
These chromoproteins can be of significant value to the other Bacillus subtilis users in the BioBrick community.


6. Most importantly: we developed a construct which makes Bacillus subtilis sense spoiled meat and produce an output in the form of a yellow or purple pigment visible by naked eye.





Our sponsors:

Retrieved from "http://2012.igem.org/Team:Groningen/Project"