Team:Groningen/Project

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[[File:Groningen2012_RR_20120713_paintpicture.png|thumb|600px|right|Fig. p1. schematic overview of the functionality of our sticker containing ''Bacillus salus carnis''.]]
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== Summary==
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Every year, ''1.300.000.000 tons'' of food are ''thrown away'' worldwide. This is one third of the global food production. One of the reasons for this is the fact that ''best before dates'' are imprecise. A reliable way of monitoring whether food is spoiled or not, could ''save upto 600 euro'' per household per year.
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</head>
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Our goal is to build a system to ''sense meat spoilage''. When a package of meat is saved after opening (1), a closed ''sticker'' containing'' Bacillus subtilis'' spores  is activated by breaking the middle compartment (2,similar to activating a glow-in-the-dark stick), whereby Alanine, and water are mixed with the spores to cause ''germination'' (3). When the germinated microbes come in contact with ''volatiles from rotting meat'', a pathway is activated which results to the production of a ''pigment''(4). When this happens, the consumer knows that he has to throw away the food (5). The different states of the volatile sensor are explained below.
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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>
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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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== States ==
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<tr>
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=== Containment of ''Bacillus subtilis'' spores: the sticker===
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<td class="accPic">
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The spores of ''Bacillus subtilis'' will be contained in a compartment which is impermeable for bacteria and liquids, but can still let through volatiles. Metabolites, water and Alanine should be separated from the spores by a breakable membrane. Mixing these compounds will cause germination.
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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>
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</table>
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<br>
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<br>
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<br>
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</body>
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</html>
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=== Germination ===
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{{Template:SponsorsGroningen2012}}
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Alanine and water can trigger germination of'' Bacillus subtilis''. The modeling team will provide information on the optimal concentrations for germination, and on the germination time.
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===Activation Pathways===
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We follow two different strategies to build a pathway in ''Bacillus subtilis'' which can react to the presence of rotten meat volatiles. One is using the TnrA pathway, which is normally expressed in absence of ammonium (reference). For the other strategy, we use a transcriptomics approach searching for promoters present in ‘’Bacillus’’ reacting to rotten meat volatiles.
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====First strategy: TnrA pathway ====
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Pre-Activation
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# The growth medium contains a low concentration of glutamine (gln), but no ammonia or ammonium (NH4).
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# Glutamine synthetase (GS) is activated due to the inadequate level of glutamine, however, it cannot synthesize more gln as it is lacking NH4.
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# The low intracellular gln level shifts the ratio of GS to feedback-inhibited GS (FBI-GS) towards a higher level of GS.
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# This higher level of GS allows a higher level of TnrA.
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# The TnrA represses the alsT promoter.
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Activation
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# Rotting meat produces NH4.
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# NH4 is enters the cell through the NrgAB ammonium transporter.
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# GS converts NH4 into gln.
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# Gln reaches the concentration required for steady cell growth.
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# The ratio of GS to FBI-GS shifts towards a higher level of FBI-GS.
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# The newly created FBI-GS binds to TnrA, creating an inactive complex.
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# TnrA is unable to repress the alsT promoter.
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# No longer repressed, alsT activates along with its positive feedback loop containing the color reporter.
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====Second strategy: identification of “PBADmeat”====
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TnrA is a repressor/activator which acts on a broad level. This makes it a tricky candidate for triggering a single reaction. Therefore, an alternative strategy is needed.
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To identify our rotten meat volatile reporter (short: PBADmeat), we performed an transcriptome analysis of ''Bacillus subtilis'' subjected to rotten meat (target) and to fresh meat (control) volatiles. Analysis of the differential expression between these two situations can lead to candidate promoters for our construct.
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For more information on the technique and some preliminary results, see the [[Team:Groningen/Wetwork|Wetwork]] page.
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===Color reporter===
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As a reporter, we choose to use a pigment. The big advantage of using a pigment over another type of reporter (for instance, GFP or an electrical signal) is that it can be detected without any equipment. A big drawback however, is the production speed of pigments.
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To make ''Bacillus subtilis'' produce more pigment, we plan to build in a positive feedback loop. Different feedback loops are available in the Registry of Standard Biological Parts (for instance PLux/LuxR and pRE/CII). We plan to characterize and - if necessary - improve one for our construct.
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We will start with using the e.chromi pigments from the iGEM team of Cambrige, 2008. These pigments (violacein and lycopene) are produced strongly in e.coli and are visible by eye at even low optical densities. To test if our construct principally works, however, we will use GFP.
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=== Death  ===
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To make sure no live GMO will get into the environment, the death of ''Bacillus'' should be reached:
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# The extracellular nutrients are depleted.
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# Color reporter builds to toxic levels.
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== Construct ==
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The standard construct is a combination of biobricks.
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*Backbone:Available in registry: BBa_K090403 (Backbone) - Single copy shuttle vector. Other option: build our own well-characterized backbone for our chassis.
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* BBa_K116632+BBa_K116602 (Positive feedback loop) - Uses the pRe promoter regulated by C11 and terminated by B0010.  Other options: BBa_J37019;BBa_K116639; BBaK116609.
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* BBa_K274100 (Reporter) - Modified carotenoid pathway to produce red pigment.
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[[File:Groningen2012_JP_20120618_pRe-CII-Lycopene_Construct_A.png|680px|center]]
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* Or, using BBa_K274002 (Reporter) - Violet pigment producer
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[[File:Groningen2012_JP_20120618_pRe-CII-Violacein_Construct_A.png|680px|center]]
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== Modeling-Labwork Cooperation ==
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Unlike previous years, there is a direct link between theory and practice.
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The modeling team of one will provide to the lab:
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# Creation, and critical analysis, of the reaction pathways from literature.
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* Identification
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# of the metabolites necessary to support growth. (Done: D-Glucose, Water, Glutamine, Potassium)
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# of the metabolites required for the reporter.
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# of the metabolites required for germination.
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# of the conditions for sporulation.
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* Quantification
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# Rates of nutrient consumption versus temperature and biomass.
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# Amount of O2 required, and the corresponding volume of air.
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# Plot of growth rate vs. temperature.
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# Plot of biomass amount vs. time
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## Superimpose curves with different starting medium concentration.
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## Indicate the points at which sporulation occurs.
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The lab will provide to the modeling team:
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# TnrA concentration as a function of extracellular glutamine concentration.
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# Growth rate as a function of extracellular glutamine concentration.
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# Rates of volatile production as a function of meat type and volume.
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== Limitations due to time constraints ==
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* We chose to make our construct at room temperature/ 37 degrees. If this project leads to  a proof of concept, a psychrotrophic bacterium like ''Bacillus cereus'' could be used as a chassis instead of ''Bacillus subtilis''. This bacterium is however a much harder to engineer and, unlike ''Bacillus subtilis'' harmful.
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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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=== 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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=== 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
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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"