Difference between revisions of "Team:Paris Bettencourt/Project/Enzyme"

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<h2 style="text-align: center">Developing a new assay platform </h2>
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<p class=text>
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Our goal in the assay group is to develop a high-throughput assay platform to test our compounds on fabric. This assay should answer the following criterias :
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<ul><li>Standardise assay : 96-well plate format</li>
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<li>Flexible for automation</li>
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<li>Low cost device</li>
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<li>Allows high-throughput screening</li>
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</ul>
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For our project we also aim to develop an open-source software to measure stain intensity of our assay platform.
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<br>We truly believe in the maker way of thinking, we try different approaches, prototype very quickly  so we can see and adjust our device. We use 3D printing, laser-cutting, but also PDMS; we first asked advice to experts and after experimenting we are really learning by doing ! 
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</p>
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Sources:<br>
 +
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<a href="https://pubchem.ncbi.nlm.nih.gov/compound/tetrachloroethylene#section=Other-Preventative-Measures">Perchloroethylene, Pubchem</a> <br>
 +
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<a href="https://toxtown.nlm.nih.gov/text_version/chemicals.php?id=22">US National Library of Medecine </a> <br>
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 +
  <a href="http://ntp.niehs.nih.gov/ntp/roc/content/listed_substances_508.pdf">Report on Carcinogens, Thirteenth edition</a> <br>
 +
 +
  <a href="http://www.developpement-durable.gouv.fr/Le-perchloroethylene-interdit-dans.html">Ministère de l'Environnement, de l'Énergie, et de la Mer</a> <br>
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<!-- <a href="http://waterhouse.ucdavis.edu/whats-in-wine">Wine composition</a> <br>-->
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  <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Assay" title="Assay">
 
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<h2 class=project>Short Summary: What We Did</h2>
 
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<br>In this project we produced synthetic enzymes to remove red wine stains from fabric. These enzymes are designed to replace perchloroethylene (PERC), a toxic solvent used in dry cleaning that will soon be banned in France.<br>
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<br>We listened to the people who will use our product, conducting face-to-face interviews with every single dry cleaner in Paris. From this came a plan for a realistic product, a stain fighting enzymatic pretreatment compatible with existing cleaning technologies and workflows.<br>
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<br>We isolated microbes capable of degrading red wine from a library of 150 strains taken from vineyard soil samples collected around the world with the help of our fellow iGEMers. The most effective microbes were submitted for whole genome resequencing, then analyzed to produce a short list of candidate stain-fighting enzymes.<br>
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<br>We modeled enzymatic activity at the fabric surface and determined that activity could be substantially improved if the enzymes had a moderate binding affinity for the fabric itself. This effectively increases the enzyme concentration at the fabric surface and reduces the quantity of enzymes lost through diffusion into the medium.<br>
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<br>We identified short amino acid sequences with affinity to cotton, linen, wool, polyester and silk using the method of phage display. The resulting Fabric Binding Domains (FBDs) were quantitiatively characterized using ELISA, to determine peptides with optimal affinity as determined by our model.<br>
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<br>We developed a high-throughput assay to quantify stain removal on real cloth. Laser-cut fabric samples are sealed to 96-well microplates and imaged on flat-bed scanners. We coded custom image analysis software to identify each circular fabric sample and measure the stain intensity.<br>
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<br>We constructed new BioBricks, fusions of our most promising fabric binding domains to our favorite wine-degrading enzymes. The resulting proteins were expressed, purified and characterized both in vitro (in solution) and in situ (on real stained fabric).<br>
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<br>Finally, we succeeded in increasing the degradation of red wine stains by XX%. These results were obtained under real world conditions, on store-bought fabrics stained with our favorite red wines. We hope these pages convince you that our project is mature, thoroughly documented and effective.<br>
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Sources:<br>
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<a href="https://pubchem.ncbi.nlm.nih.gov/compound/tetrachloroethylene#section=Other-Preventative-Measures">Perchloroethylene, Pubchem</a> <br>
 
  
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<a href="https://toxtown.nlm.nih.gov/text_version/chemicals.php?id=22">US National Library of Medecine </a> <br>
 
−
 
−
  <a href="http://ntp.niehs.nih.gov/ntp/roc/content/listed_substances_508.pdf">Report on Carcinogens, Thirteenth edition</a> <br>
 
−
 
−
  <a href="http://www.developpement-durable.gouv.fr/Le-perchloroethylene-interdit-dans.html">Ministère de l'Environnement, de l'Énergie, et de la Mer</a> <br>
 
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Revision as of 21:02, 11 October 2016


Developing a new assay platform

Our goal in the assay group is to develop a high-throughput assay platform to test our compounds on fabric. This assay should answer the following criterias :

  • Standardise assay : 96-well plate format
  • Flexible for automation
  • Low cost device
  • Allows high-throughput screening
For our project we also aim to develop an open-source software to measure stain intensity of our assay platform.
We truly believe in the maker way of thinking, we try different approaches, prototype very quickly so we can see and adjust our device. We use 3D printing, laser-cutting, but also PDMS; we first asked advice to experts and after experimenting we are really learning by doing !

Sources:
Perchloroethylene, Pubchem
US National Library of Medecine
Report on Carcinogens, Thirteenth edition
Ministère de l'Environnement, de l'Énergie, et de la Mer

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com
2016.igem.org