Team:Groningen/DesignTest2

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<z1>Collaboration</z1><br>
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<z1>Abstract</z1>
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<font color=#FF6700><b>Characterization of  <i>Bacillus subtilis</i> plasmid  backbone created by  iGEM LMU Munich 2012</b></font> <br>
 
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Munich team has developed a plasmid backbone that is suitable for gene expression in <i>B. subtilis</i> <b>(Bba_K823023)</b>.
 
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This plasmid backbone contains an integration locus in amyE, which means after being transformed into <i>B. subtilis</i>,
 
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the cloned insert will be integrated with <i>B. subtilis</i> genome at the amyE locus. <i>B. subtilis</i> is known for its
 
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starch degrading ability and amyE is a gene responsible for the starch degradation phenotype in <i>B.subtilis</i> by regulating a-amylase production.
 
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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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</table>
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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
 +
a pigment coding gene. This promoter, <z7>identified by microarray analysis</z7>, is significantly upregulated in the presence of
 +
<z7>volatiles from spoiling meat</z7>. The activity of the <z7>promoter</z7> regulates the expression of the <z7>pigment reporter</z7> 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
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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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We characterized this plasmid backbone by doing a simple growth experiment on a starch agar plate. <i>B. subtilis</i>
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strains (168 wildtype, pSac-Cm, and K823023, clockwise respectively) were inoculated in LB agar+0.4% starch and incubated overnight
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in 37<sup>o</sup>C (left picture). After the addition of 0.1% iodine, the clear zone was formed around <i>B. subtilis</i>
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wildtype and <i>B. subtilis</i> strain containing pSac-Cm. There was no clear zone observed around
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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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<i>B. subtilis</i> strain containing backbone K823023 (right picture).
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<br>
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<br>
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The inability of the <i>B. subtilis</i> strain containing BBa_K823023 to degrade starch was simply demonstrating that the amyE locus in
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the <i>B. subtilis</i> genome had been replaced. Apart from this characterization experiment, our team has been using BBa_K823023
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as a plasmid backbone for our volatile detection device in <i>B. subtilis</i> (link to sboA-lycopene page).
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<br>
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<br>
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<z4>References</z4>
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<ol class="ref">
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<li>Nicholson, W.L, Park, Y.K, Henkin, T.M, Won, M., Weickert, M.J, Gaskell, J.A, Chambliss, G.H. 2006. Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence. J Mol Biol. 1987 Dec 20;198(4):609-18.</li>
+
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<li>Zeigler, Daniel R. 2002. Bacillus Genetic Stock Center Catalog of Strains, Seventh Edition, Volume 4: Integration Vectors for Gram-Positive Organisms. The Bacillus Genetic Stock Center. Department of Biochemistry, The Ohio State University. 484 West Twelfth Avenue,Columbus, Ohio 43210.</li>
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<br>
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<p class="margin">
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We also filled in their survey and now very proud that we deserved the Bavarian Medal.<br>
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</p>
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<br>
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<p class="margin"><FONT COLOR=#ff6700><b>iGEM LMU Munich</b></FONT><br><br>
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This iGEM team is also working on <i>B. subtilis</i> and therefore an ideal partner for testing each other’s constructs.
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They sent us 6 plasmids and 4 promoters and we are testing them for characterization. Hopefully we are able to return our constructs to them soon!</p>
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<p class="margin"><FONT COLOR=#ff6700><b>iGEM Uppsala</b></FONT><br><br>
 
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At the beginning of August we received the chromoproteins amilCP (blue), amilGFP (yellow) from
 
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<A HREF="https://2011.igem.org/Team:Uppsala-Sweden" TARGET="_blank"><FONT COLOR=#ff6700>iGEM Uppsala 2011</FONT></A>.
 
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We were able to transform their BBa_K592009 and BBa_K592010 behind our identified promoters into <i>E.coli</i>.
 
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For now we are waiting untill they are transformed in <i>B.subtilis</i>, to check if the blue and yellow color also works in this organism.
 
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For the latest results, check our <A HREF="https://2012.igem.org/Team:Groningen/Pigments" TARGET="_blank"><FONT COLOR=#ff6700>Pigment page</FONT></A>.
 
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<p class="margin"><FONT COLOR=#ff6700><b>Dutch iGEM teams (iGEM Wageningen, Amsterdam, Delft, Eindhoven)</b></FONT><br><br>
 
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All of the Dutch iGEM teams collaborated to create an iGEM presence at the
 
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<A HREF="http://www.discoveryfestival.nl/" TARGET="_blank"><FONT COLOR=#ff6700>Discovery Festival</FONT></A> on Friday 28 September
 
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in Amsterdam, Rotterdam and Eindhoven. We will aim to get the public in touch with Synthetic Biology in a playful way.
 
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<br>
 
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<br>
 
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Besides these Dutch iGEM meetings and the Discovery Festival, we also collaborated with Wageningen and Amsterdam on a molecular level.
 
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Many times our transformation of <i>E.coli</i> failed, so we ran out of the backbone biobricks for <i>B. subtilis</i>. Luckily Wageningen
 
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and Amsterdam were so kind to provide us with some of their bricks. Thank you guys!
 
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<br>
<br>
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<p class="margin"><FONT COLOR=#ff6700><b>iGEM Calgary 2012 </b></FONT><br><br>
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<div class="cte2">
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Emily Hicks from the winning Calgary iGEM team in 2011, and now member of iGEM Calgary 2012, has studied in Groningen during her exchange period.  
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<div class="ctd2">
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She was very interested in her iGEM Groningen colleagues; therefore we asked her to give a lecture about her previous iGEM experience.  
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<a name="MainAcc"></a><z2 >Our main accomplishments</z2><br>
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We were really motivated by Emily's tips concerning medal requirements, information about wiki etc. Thank you Emily!
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<br><br>
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<p class=orange>In the lab</p>
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<p class="marginleft">
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<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> Most importantly: we developed a construct which makes <i>Bacillus subtilis</i> sense spoiled meat and produce an output in the form of a yellow pigment visible by naked eye.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> Design of the “<a href="https://2012.igem.org/Team:Groningen/Sticker">Sticker</a>”: semi-permeable capsule: bacteria are kept inside, volatiles can go through. Proof that <i>Bacillus subtilis</i> grows inside the sticker.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> Development of BioBrick psac-cm for <i>Bacillus subtilis</i>. <br><a href="http://partsregistry.org/Part:BBa_K818000" target=_blank><font size=4 color=#FF6700><b>Easy cloning in <i>Bacillus subtilis</i> in Biobrick fashion for the first time!</b></font></a> Advantages: easy to check, BioBrick compatible, <i>E. coli</i> compatible, stable insertion into <i>Bacillus subtilis</i> chromosome.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> Identification of spoiled meat sensors by transcriptome analysis. Usage of an innovative experimental setup which can be used to test more sensors in the future.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> We made AmilCP and AmilGFP be expressed in <i>Bacillus subtilis</i>. These chromoproteins can be of significant value to the other <i>Bacillus subtilis</i> users in the BioBrick community.<br><br><br>
</p>
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<p class=orange>Other</p>
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<p class="marginleft">
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<p class="margin"><FONT COLOR=#ff6700><b> iGEM Virginia</b></FONT><br><br>
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<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> Smart spores activation system: inspired by "glow in the dark stick", we designed a sticker with two compartments. One compartment holds the spores, the other the nutrients; when the nutrients compartment is braked, the spores start to germinate, activating the sticker.<br><br>
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Prof. Papin, supervisor to the iGEM Virginia team and supervising author to the idFBA article, graciously shared his knowledge and scripts relating to
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<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> We explored the definition of spoiled meat and different ways to test this.<br><br>
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idFBA.
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<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> We identified various compounds present in spoiled meat by Gas Chromatography - Mass Spectrometry.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2012/d/df/IGEMGroningen2012_AD20120915_Tick.png" width=20> We made a model which gives insight on how to tweak the growth of <i>Bacillus subtilis</i> based upon literature, experimental data and flux balance analysis.<br>
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{{Template:SponsorsGroningen2012}}

Latest revision as of 00:16, 27 September 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.



Our main accomplishments


In the lab

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

Design of the “Sticker”: semi-permeable capsule: bacteria are kept inside, volatiles can go through. Proof that Bacillus subtilis grows inside the sticker.

Development of BioBrick psac-cm for Bacillus subtilis.
Easy cloning in Bacillus subtilis in Biobrick fashion for the first time! Advantages: easy to check, BioBrick compatible, E. coli compatible, stable insertion into Bacillus subtilis chromosome.

Identification of spoiled meat sensors by transcriptome analysis. Usage of an innovative experimental setup which can be used to test more sensors in the future.

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


Other

Smart spores activation system: inspired by "glow in the dark stick", we designed a sticker with two compartments. One compartment holds the spores, the other the nutrients; when the nutrients compartment is braked, the spores start to germinate, activating the sticker.

We explored the definition of spoiled meat and different ways to test this.

We identified various compounds present in spoiled meat by Gas Chromatography - Mass Spectrometry.

We made a model which gives insight on how to tweak the growth of Bacillus subtilis based upon literature, experimental data and flux balance analysis.


Our sponsors:

Retrieved from "http://2012.igem.org/Team:Groningen/DesignTest2"