Difference between revisions of "Team:Paris Bettencourt/Results"

 
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<div id=subheader>
 
<div id=subheader>
 
<div style="margin-top:20px; margin-bottom:20px">
 
<div style="margin-top:20px; margin-bottom:20px">
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<img src="https://static.igem.org/mediawiki/2016/1/19/Paris_Bettencourt-Award.jpg" height="300px"/>
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<div style="margin-top:20px; margin-bottom:40px">
 +
 
<div class="panel" >
 
<div class="panel" >
 
  <a href="https://2016.igem.org/Team:Paris_Bettencourt/Results" title="Results">
 
  <a href="https://2016.igem.org/Team:Paris_Bettencourt/Results" title="Results">
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<div id=definition>
 
<div id=definition>
−
<h2>Achievements</h2>
+
<h2>Results</h2>
 
<p>  
 
<p>  
  
−
<h2 class="red">Automated and DIY microplate on fabric</h2>
+
<h2 class="red">Automated/DIY microplate on fabric-Image processing tool for stain measurement</h2>
 
  <p class=”input”>  
 
  <p class=”input”>  
 
An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles.  
 
An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles.  
 
<br>
 
<br>
−
1. Spread a flat, wet cotton sheet on glass substrate of 96 well plate dimension.  
+
<ul>
 +
<li> Spread a flat, wet cotton sheet on glass substrate of 96 well plate dimension.  
 +
 
 +
<li> 96 Wet cotton circles of 6.5mm are laser cut on glass substrate.
 +
 
 +
<li> Remove the fabric. The circles of cotton stay on the glass substrate.
 +
 
 +
<li> Place the glass substrate of cotton circles on fluorescent microplate such that all circles face the wells and align well.
 +
 
 +
<li> Hold it tight and move the glass substrate laterally so that all the cotton circles fall into the wells.        
 +
 
 +
<li> Put the tip plate holder on the top of the microplate and adhere it to the microplate using a glue.
 +
 
 +
<li> Cover it with a lid and the microplate is ready to use for experiments. The same can be done for other fabrics like silk, polystyrene, nylon etc.
 +
 
 +
</ul>
 +
An Image processing plugin was developed to measure the pixel intensity of each well of the scanned image of Microplate on fabric. The relative change in pixel intensity is the quantitative measure for degradation of stain.
 +
       
 
<br>
 
<br>
−
2. 96 Wet cotton circles of 6.5mm are laser cut on glass substrate.
 
 
<br>
 
<br>
−
3. Remove the fabric. The circles of cotton stay on the glass substrate.
+
<div style="text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/2016/e/ea/Paris_Bettencourt-Result.png" alt="Microplate with lasercut fabric" style="width:500px;height:375px;">
 +
</p>
 
<br>
 
<br>
−
4. Place the glass substrate of cotton circles on fluorescent microplate such that all circles face the wells and align well.
 
 
<br>
 
<br>
−
5. Hold it tight and move the glass substrate laterally so that all the cotton circles fall into the wells.          
+
 
 +
 
 +
<h2 class="red">Selected Peptides from Binding Domain Group</h2>
 +
 +
    <p>
 +
    Phage display yields peptides binding cotton, linen, wool, polyester and silk. <br>
 +
 +
The phage library was panned against fabric samples of cotton, linen, wool, polyester and silk. The cycle of phage binding, elution, and amplification was repeated three times for each fabric. Following enrichment, individual clones were isolated and sequenced to determine the sequence of their displayed peptide. We isolated and sequenced at least 40 phage plaques from each fabric.<br>
 +
 +
   
 +
          <div style="text-align:center;">
 +
                     
 +
 
 +
     
 +
 
 +
</div>     
 +
</div>
 +
    <br>
 +
<p>
 +
 +
The nine sequences out of 40 FBD peptides were selected  for validation with ELISA.
 +
        <div id="figurebox">
 +
        <div style="text-align:center;">
 +
        <img src="https://static.igem.org/mediawiki/2016/4/40/Paris_Bettencourt-Elisa_validation.jpg" alt="Paris_bettencourt-Selected_ELISA_Validation" style ="width:700px;height:400px;">
 +
        </p>
 +
        </div>
 +
        </div>
 +
          
 
<br>
 
<br>
−
5. Put the tip plate holder on the top of the microplate and adhere it to the microplate using a glue.
+
    <br>
 +
    <br>
 +
   
 +
   
 +
<h2 class="red">Catechol dioxydases were expressed with and without Fabric Binding Domains and their functionality was demonstrated in both cases</h2>
 +
<p class=”input”>
 +
Six candidate plant and bacterial enzymes were tested for expression in <i>E. coli</i>
 
<br>
 
<br>
−
6. Cover it with a lid and the microplate is ready to use for experiments. The same can be done for other fabrics like silk, polystyrene, nylon etc.
+
Three out of six enzymes were functionally expressed in <i>E. coli</i> and tested for their ability to degrade their natural substrate
−
+
<br>
 +
Feasibility of fusing proteins with FBDs was tested by fusing them to GFP and assessing their GFP ability and their ability to bind to several fabrics
 +
<br>
 +
The selected enzymes were fused with Fabric Binding Domains and their activity was still observed
 +
 
 +
 
 
         
 
         
 
<br>
 
<br>
 
<br>
 
<br>
−
<img class="assay" src="https://static.igem.org/mediawiki/2016/b/b4/Paris_Bettencourt-Notebook_Assay_Finaldesign_topview.jpg" alt=« Success" height=“150px“ />
+
<div style="text-align:center;">
−
<img class="assay" src="https://static.igem.org/mediawiki/2016/1/1e/Paris_Bettencourt-Notebook_Assay_Finaldesign_topview2.jpg" alt=« Success" height=“150px“ />
+
<img src="https://static.igem.org/mediawiki/2016/5/5f/Paris_Bettencourt-enzyme_BOTHONONE.jpg" style="width:500px;height:375px;">
−
<img class="assay" src="https://static.igem.org/mediawiki/2016/7/75/Paris_Bettencourt-Notebook_Assay_Finaldesign_Dilutiontest.jpg" alt=« Success" height=“150px“ />
+
−
<img class="assay" src="https://static.igem.org/mediawiki/2016/d/d6/Paris_Bettencourt-Notebook_Assay_Finaldesign_beforedilution.jpg" alt=« Success" height=“150px“ />
+
−
<img class="assay" src="https://static.igem.org/mediawiki/2016/4/48/Paris_Bettencourt-Notebook_Assay_Finaldesign_afterdilution.jpg" alt=« Success" height=“150px“ />
+
 
</p>
 
</p>
 
<br>
 
<br>
 
<br>
 
<br>
−
<h2 class="red">Selected Peptides from Binding Domain Group</h2>
+
 
−
<p class=”input”>  
+
 
−
From approximately 20 sequences for 5 different fabrics each we picked total of 9 peptide sequence to pass on to Enzyme search group to fuse it with enzymes they are working on and study their efficiency in binding fabrics.<br>
+
<h2 class="red">Microbes that degrade indigo </h2>
−
The peptides are named FBD 1-9 for Fabric binding domain - 1 to 9.<br>
+
 
−
Out of the 9 sequences 5 are specific, i.e, one sequence specific for each fabric. The sequences were not selected based on the consensus sequence but their repeatition in that particular fabric or some unique feature in the peptide. and the other 4 sequences are non specific, found in multiple fabrics.<br>
+
<p class="input">We succeeded in finding 16 microbes that grow on denim, covered with M9 media. <br>After two rounds of culture in liquid media with indigo and measurements of absorbance as a way to assess indigo degradation, we found 3 strains highly capable at degrading indigo : <i>Streptmoyces fumigatiscleroticus </i>, <i> Streptomyces coelicolor </i> and <i> Pantoea agglomerans </i>. </p>
−
<br>
+
<img src="https://static.igem.org/mediawiki/2016/8/8f/Paris_Bettencourt-File_Denim.jpg" alt="denim sample from bottle" alt="Microbes on denim" width="300px"/>
 +
<br><br>
 +
<h2 class="red">Bpul laccase degrades indigo </h2>
 +
<div style="text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/2016/4/4a/T--Paris_Bettencourt--indigo_group_fig4.jpg" width=700px>
 +
</div>
 +
<p>
 +
Structural formulas of two substrates (indigo carmine, and indigo) of Bpul laccase are showing structural similarity. Since it has been shown that bpul degrades indigo carmin, we went to prove its ability to degrade indigo. All results shown in the figure above are made with our bpul gene expressed in the E. coli strain BL21(DE3). The protein gel (figure above B) is showing an over-expression of the bpul, after the induction with IPTG. When comparing uninduced bpul sample with the control sample (BL21(DE3)) we can notice leakiness of our promoter, which is probably due to great transcriptional strength of T7 polymerase. In the degradation experiments (figure above C and D), both the transformant BL21(DE3)-bpul, and the negative control BL21(DE3) have been induced with IPTG. The cell extracts of both strains were tested for the ability to degrade indigo carmine and indigo. 10µM acetosyringone (ACS) was added to the reaction mixture to facilitate the oxidation of the substrates. ACS is a known mediator of the laccases and it is often used and needed in the studies of the  laccase activitiy As expected, the 1mM indigo carmine solution is entirely degraded within the 13 hours of the experiment in the cell extract which contains overexpressed bpul. Additionally, we have shown that more than 60% of indigo can be degraded by bpul laccase in the 13 hours of the experiment. It is important to notice, that in longer experiments would probably lead to even more degradation since the degradation curve didn't reach the saturation.
 
</p>
 
</p>
−
<ul>
 
−
    <li>
 
−
        FBD1 -  Is non specific, it is repeated in all 5 fabrics.<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/7/72/Paris_Bettencourt-FBD1.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_1"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD 2, 3, 4- Repeated in all fabric. <br>
 
−
       
 
−
        FBD2-
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/e/ea/Paris_Bettencourt-FBD2.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_2"/>
 
−
        </div>
 
−
        FBD3-
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/d/d0/Paris_Bettencourt-FBD3.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_3"/>
 
−
        </div>
 
−
        FBD4-
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/b/b7/Paris_Bettencourt-FBD4.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_4"/>
 
−
        </div>
 
−
    <li>
 
−
        FBD5 - Specific to Wool, it was repeated 3 times out of 16 sequences.<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/3/3a/Paris_Bettencourt-FBD5.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_5"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD6 - Specific to cotton<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/7/7a/Paris_Bettencourt-FBD6.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_6"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD7 - Specific to Silk<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/8/84/Paris_Bettencourt-FBD7.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_7"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD8 - Specific to Linen, the sequence was selected because it has multiple proline residues<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/9/9c/Paris_Bettencourt-FBD8.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_8"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD9 - Specific to polyester, it was selected because of its high number of positively charged amino acids<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/9/98/Paris_Bettencourt-FBD9.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_9"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FBD10 - Picked from literature, Guo et.al, (2013) it will serve as a positive control - shown to bind to cellulose<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/5/58/Paris_Bettencourt-FBD10.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_10"/>
 
−
        </div>
 
−
    </li>
 
−
    <li>
 
−
        FDB11 - Random amino acids put together(7aa) to be used as negative control.<br>
 
−
        <div style="text-align:center;">
 
−
            <img src="https://static.igem.org/mediawiki/2016/e/ee/Paris_Bettencourt-FBD11.png" alt="Paris_bettencourt-Selected_Fabric_Binding_Domain_11"/>
 
−
        </div>
 
−
    </li>
 
  
−
</ul>
+
<br><br>
 +
 
 +
<h2 class="red">Construction of the strain database by the microbiology team</h2>
 +
<p>
 +
This summer, our group designed an experiment to find the best stain fighting enzymes that nature has to offer.
 +
We build a big database with 186 strains through selective and non-selective plating on different media.
 +
<ul>
 +
            <li>We managed to isolate species from all around the world through iGEM collaborations.</li>
 +
            <li>186 bacteria were tested for quercetin degradation.</li>
 +
            <li>A 186 bacterial database was built and characterized.</li>
 +
<li>20 strains produced more than 50% quercitin degradation.</li>
 +
            <li>A phylogenetic tree of 174 different  bacterial species was created .</li>
 +
            <li>3 bacterias were shown able to degrade anthocyanin</li>
 +
            <li>Common candidate genes were selected through genome sequencing of 4 different bacterial strains.</li>
 +
      </ul>
 +
<br><br>
 +
 
 +
<center><img src="https://static.igem.org/mediawiki/2016/d/dc/Paris_Bettencourt-Phylogenetic_Tree.jpg" alt="TOL" alt="Microbes on denim" width="1100px"/></center>
 +
<br><br>
 +
<center><img src="https://static.igem.org/mediawiki/2016/1/1f/Paris_Bettencourt-File_Quercetin_.jpg" alt="Quercetin degradation" alt="Microbes on denim" width="1100px"/></center>
 +
</p>
 +
<div style="margin-top:20px; margin-bottom:20px">
 +
<div class="panel" >
 +
<a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Assay" title="Assay">
 +
<div id="pspanel" class="subpanel8"  onmouseover="chgtrans(this)">
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/b/b9/Paris_Bettencourt-Assay_Button2.png" width="150px" height="250px"/>
 +
  <div class="titlebox">
 +
    <center><img src="https://static.igem.org/mediawiki/2016/e/e8/Paris_Bettencourt-Logo_Assay.png" style="height:60px;"/></center>
 +
    <div style="width:60%;margin-left:20%;margin-bottom:20px;"><hr></div>
 +
    Assay
 +
  </div>
 +
</div>
 +
      </a>
 +
      <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Microbiology" title="Microbiology">
 +
<div id="dspanel" class="subpanel8">
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/e/e0/Paris_Bettencourt-Microbiology_Button2.png" width="150px" height="250px"/>
 +
  <div class="titlebox">
 +
    <center><img src="https://static.igem.org/mediawiki/2016/f/f8/Paris_Bettencourt-Logo_Microbiology.png" style="height:60px;"/></center>
 +
    <div style="width:60%;margin-left:20%;margin-bottom:20px;"><hr></div>
 +
    Microbiology
 +
  </div>
 +
</div>
 +
      </a>
 +
      <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Enzyme" title="Enzyme">
 +
<div id="tcpanel" class="subpanel8">
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/d/d0/Paris_Bettencourt-Enzyme_Button2.png" width="150px" height="250px"/>
 +
  <div class="titlebox">
 +
    <center><img src="https://static.igem.org/mediawiki/2016/9/9d/Paris_Bettencourt-Logo_Enzyme.png" style="height:60px;"/></center>
 +
    <div style="width:60%;margin-left:20%;margin-bottom:20px;"><hr></div>
 +
  Enzyme
 +
  </div>
 +
</div>
 +
      </a>
 +
      <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Binding" title="Binding domains">
 +
<div id="thpanel" class="subpanel8">     
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/6/68/Paris_Bettencourt-Binding_Button2.png" width="150px" height="250px"/>
 +
  <div class="titlebox">
 +
    <center><img src="https://static.igem.org/mediawiki/2016/4/44/Paris_Bettencourt-Logo_Binding.png" style="height:60px;"/></center>
 +
    <div style="width:60%;margin-left:20%;margin-bottom:20px;"><hr></div>
 +
    Binding
 +
  </div>
 +
</div>
 +
      </a>
 +
    <a href="https://2016.igem.org/Team:Paris_Bettencourt/Project/Indigo" title="Indigo">
 +
<div id="thpanel" class="subpanel8">     
 +
  <img class="narrowimg" src="https://static.igem.org/mediawiki/2016/7/75/Paris_Bettencourt-Indigo_Button2.png" width="150px" height="250px"/>
 +
  <div class="titlebox">
 +
    <center><img src="https://static.igem.org/mediawiki/2016/f/f0/Paris_Bettencourt-Logo_Indigo.png" style="height:60px;"/></center>
 +
    <div style="width:60%;margin-left:20%;margin-bottom:10px;"><hr></div>
 +
    Indigo
 +
  </div>
 +
</div>
 +
      </a>
 +
<a href="https://2016.igem.org/Team:Paris_Bettencourt/Model" title="Model">
 +
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Latest revision as of 23:27, 1 December 2016


Results
Medal Requirements
Parts
Interlab Study

Results

Automated/DIY microplate on fabric-Image processing tool for stain measurement

An automated 96 well microplate for fabric was made using a fluorescent microplate, 96 well tip holder and laser-cut cotton circles.

  • Spread a flat, wet cotton sheet on glass substrate of 96 well plate dimension.
  • 96 Wet cotton circles of 6.5mm are laser cut on glass substrate.
  • Remove the fabric. The circles of cotton stay on the glass substrate.
  • Place the glass substrate of cotton circles on fluorescent microplate such that all circles face the wells and align well.
  • Hold it tight and move the glass substrate laterally so that all the cotton circles fall into the wells.
  • Put the tip plate holder on the top of the microplate and adhere it to the microplate using a glue.
  • Cover it with a lid and the microplate is ready to use for experiments. The same can be done for other fabrics like silk, polystyrene, nylon etc.
An Image processing plugin was developed to measure the pixel intensity of each well of the scanned image of Microplate on fabric. The relative change in pixel intensity is the quantitative measure for degradation of stain.

Microplate with lasercut fabric



Selected Peptides from Binding Domain Group

Phage display yields peptides binding cotton, linen, wool, polyester and silk.
The phage library was panned against fabric samples of cotton, linen, wool, polyester and silk. The cycle of phage binding, elution, and amplification was repeated three times for each fabric. Following enrichment, individual clones were isolated and sequenced to determine the sequence of their displayed peptide. We isolated and sequenced at least 40 phage plaques from each fabric.


The nine sequences out of 40 FBD peptides were selected for validation with ELISA.

Paris_bettencourt-Selected_ELISA_Validation




Catechol dioxydases were expressed with and without Fabric Binding Domains and their functionality was demonstrated in both cases

Six candidate plant and bacterial enzymes were tested for expression in E. coli
Three out of six enzymes were functionally expressed in E. coli and tested for their ability to degrade their natural substrate
Feasibility of fusing proteins with FBDs was tested by fusing them to GFP and assessing their GFP ability and their ability to bind to several fabrics
The selected enzymes were fused with Fabric Binding Domains and their activity was still observed



Microbes that degrade indigo

We succeeded in finding 16 microbes that grow on denim, covered with M9 media.
After two rounds of culture in liquid media with indigo and measurements of absorbance as a way to assess indigo degradation, we found 3 strains highly capable at degrading indigo : Streptmoyces fumigatiscleroticus , Streptomyces coelicolor and Pantoea agglomerans .

denim sample from bottle

Bpul laccase degrades indigo

Structural formulas of two substrates (indigo carmine, and indigo) of Bpul laccase are showing structural similarity. Since it has been shown that bpul degrades indigo carmin, we went to prove its ability to degrade indigo. All results shown in the figure above are made with our bpul gene expressed in the E. coli strain BL21(DE3). The protein gel (figure above B) is showing an over-expression of the bpul, after the induction with IPTG. When comparing uninduced bpul sample with the control sample (BL21(DE3)) we can notice leakiness of our promoter, which is probably due to great transcriptional strength of T7 polymerase. In the degradation experiments (figure above C and D), both the transformant BL21(DE3)-bpul, and the negative control BL21(DE3) have been induced with IPTG. The cell extracts of both strains were tested for the ability to degrade indigo carmine and indigo. 10µM acetosyringone (ACS) was added to the reaction mixture to facilitate the oxidation of the substrates. ACS is a known mediator of the laccases and it is often used and needed in the studies of the laccase activitiy As expected, the 1mM indigo carmine solution is entirely degraded within the 13 hours of the experiment in the cell extract which contains overexpressed bpul. Additionally, we have shown that more than 60% of indigo can be degraded by bpul laccase in the 13 hours of the experiment. It is important to notice, that in longer experiments would probably lead to even more degradation since the degradation curve didn't reach the saturation.



Construction of the strain database by the microbiology team

This summer, our group designed an experiment to find the best stain fighting enzymes that nature has to offer. We build a big database with 186 strains through selective and non-selective plating on different media.

  • We managed to isolate species from all around the world through iGEM collaborations.
  • 186 bacteria were tested for quercetin degradation.
  • A 186 bacterial database was built and characterized.
  • 20 strains produced more than 50% quercitin degradation.
  • A phylogenetic tree of 174 different bacterial species was created .
  • 3 bacterias were shown able to degrade anthocyanin
  • Common candidate genes were selected through genome sequencing of 4 different bacterial strains.


TOL


Quercetin degradation


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