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

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<h2 class="red">Selected Peptides from Binding Domain Group</h2>
 
<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>
+
    <p>
The peptides are named FBD 1-9 for Fabric binding domain - 1 to 9.<br>
+
    Phage display yields peptides binding cotton, linen, wool, polyester and silk. <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>
+
<br>
+
Our phage display experiments were performed with the commercial Ph.D.-7 Phage Display Peptide Library (NEB # E8100S). This library consists 100 copies each of 109 phages expressing a randomized 7-mer peptide fused to the phage N-terminal pIII coat protein. The displayed peptide is separated by a short Gly-Gly-Gly-Ser linker to minimize interactions between the displayed peptide and the phage itself.<br> <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 (Table 1).<br>
 +
 +
    <div id="figurebox">
 +
<div style="text-align:center;">
 +
                        <table style="width:90%">
 +
                        <tr>
 +
                            <th align="center">COTTON</th>
 +
                            <th align="center">LINEN</th>
 +
                            <th align="center">SILK</th>
 +
                            <th align="center">WOOL</th>
 +
                            <th align="center">POLYESTER</th>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">GVLRYAP</td>
 +
                            <td align="center">STNPTSL</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">ADIRHIK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">RLLQYNS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">YMGPSKT</td>
 +
                            <td align="center">MPRLPPH</td>
 +
                        </tr>
 +
           
 +
                        <tr>
 +
                            <td align="center">GVKSEQL</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">QFDHWRN</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">WHLPAQR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">AQSNPKN</td>
 +
                            <td align="center">MRLSVPN</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ELAGTTW</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">KNANSRE</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">MSNTLDP</td>
 +
                            <td align="center">SLLTHNM</td>
 +
                            <td align="center">GKNLMNM</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">ADARYKS</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">VFQTTYK</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ISTTLFP</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">ASSHIHH</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">TTHPRWG</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">PSNRQNT</td>
 +
                            <td align="center">GASNIWN</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">SFLVTRN</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">KTAMKGP</td>
 +
                            <td align="center">SILPVTR</td>
 +
 
 +
                            <td align="center">GASNIWN</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">TSNRAPY</td>
 +
                            <td align="center">GSTSFSK</td>
 +
                            <td align="center">ISTTLFP</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">ALANFEP</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">GALAKDE</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">MRLSVPN</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">MRLSVPN</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">XPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
   
 +
                  <tr>
 +
                            <td align="center">ERGFLLL</td>
 +
                            <td align="center">QPIYRVQ</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">SIHERAK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">QFDHWRN</td>
 +
                            <td align="center">ADARYKS</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">VECINNC</td>
 +
                            <td align="center">WTNVFVG</td>
 +
                            <td align="center">DETCSSM</td>
 +
                            <td align="center">GQSVVSL</td>
 +
                            <td align="center">ADARYKS</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">XPRLPPX</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">HWNTVVS</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">HYPPVDD</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">METVVSS</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">XPXXPPT</td>
 +
                            <td align="center">XPRLPPA</td>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">MQEMRQM</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">SNYHWRM</td>
 +
                        </tr>
 +
   
 +
                        <tr>
 +
                            <td align="center">TSDATQR</td>
 +
                            <td align="center">XPRLXPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">NNSVSMN</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">HNWMHQN</td>
 +
                            <td align="center">MPRLPPX</td>
 +
                            <td align="center">FPSPMVG</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">SILPVTR</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">AWPYVTL</td>
 +
                            <td align="center">MRLSVPN</td>
 +
                            <td align="center">MPRLPPA</td>
 +
   
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">AQSNPKN</td>
 +
                            <td align="center">HDSPTAA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">TDHAHRY</td>
 +
                            <td align="center">ASPDQEK</td>
 +
                            <td align="center">KNANSRE</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">FRKKRKS</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">SVVMPHG</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">TESAPTL</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">QFPPPPG</td>
 +
                            <td align="center">MPRLPPX</td>
 +
                            <td align="center">ADIRHIK</td>
 +
                            <td align="center">SLETMSN</td>
 +
                        </tr>
 +
                          <tr>
 +
                            <td align="center">FSRSNNT</td>
 +
                            <td align="center">HDSPTAA</td>
 +
                            <td align="center">MPRLPPX</td>
 +
                            <td align="center">YMGPSKT</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
   
 +
                        <tr>
 +
                            <td align="center">TDMTAPK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">HDVMWQR</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">MTQQLHT</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">SSHSVQR</td>
 +
                            <td align="center">TNLHINP</td>
 +
                            <td align="center">HWNTVVS</td>
 +
                            <td align="center">TVHVHKT</td>
 +
                            <td align="center">VPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">GLHYDHS</td>
 +
                            <td align="center">AGHVVPR</td>
 +
                            <td align="center">MLQGNGY</td>
 +
                            <td align="center">ADIRHIK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
 
 +
                        <tr>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">HWNTVVS</td>
 +
                            <td align="center">ASSHIHH</td>
 +
                            <td align="center">YMGPSKT</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center">DPRLSPT</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">GSTSFSK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">QGDYFTY</td>
 +
                            <td align="center">TLINYRG</td>
 +
                            <td align="center">AGHVVPR</td>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">VTLPDPR</td>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">TRPTDTI</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">VTLPDPR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">MPRXPPA</td>
 +
                            <td align="center">GSTSFSK</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">FSRSNNT</td>
 +
                            <td align="center">SILPVXR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">HDSPTAA</td>
 +
                            <td align="center"> </td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">ADARYKS</td>
 +
                            <td align="center">HDVMWQR</td>
 +
                            <td align="center">MPRLPPA</td>
 +
                            <td align="center">YMGPSKT</td>
 +
                            <td align="center"> </td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center">SILPVTR</td>
 +
                            <td align="center"> </td>
 +
                            <td align="center"> </td>
 +
                            <td align="center"> </td>
 +
                            <td align="center"> </td>
 +
                        </tr>
 +
                        </table>
 +
    <p>
 +
<b>Table 1A.</b> Novel fabric binding domains with diverse properties.
 
</p>
 
</p>
<ul>
+
</div>
    <li>
+
         <br>
        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;">
 +
                        <table style="width:90%">
 +
                        <tr>
 +
                            <th align="center">COTTON</th>
 +
                            <th align="center">LINEN</th>
 +
                            <th align="center">SILK</th>
 +
                            <th align="center">WOOL</th>
 +
                            <th align="center">POLYESTER</th>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center"> <font color="#8B1813">FSRSNNT(2)</font> </td>
 +
                            <td align="center" rowspan="2"><font color="#438CEE"> SILPVTR(2)</font> </td>
 +
                            <td align="center"><font color="#183255">AQSNPKN(2)</font> </td>
 +
                            <td align="center"> <font color="#183F24">MRLSVPN(2)</font> </td>
 +
                            <td align="center" rowspan="2"> <font color="#138B3F">ADARYKS(3)</font></td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td align="center"><font color="#F6403A"> MPRLPPA(2)</font> </td>
 +
                           
 +
                            <td align="center"><font color="#8C998D"> AWPYVTL(2)</font></td>
 +
                            <td align="center"> <font color="#FF33E3">ASSHIHH(3)</font></td>
 +
                           
 +
                        </tr>
 +
           
 +
                        <tr>
 +
                            <td align="center"> <font color="#FF33E3">ASSHIHH(4)</font></td>
 +
                            <td align="center" rowspan="3"><font color="#F6403A"> MPRLPPA(25)</font></td>
 +
                            <td align="center"> <font color="#08807A">KNANSRE(2)</font></td>
 +
                            <td align="center"> <font color="#138B3F">ADARYKS(6)</font></td>
 +
                            <td align="center" rowspan="3"><font color="#F6403A"> MPRLPPA(20)</font></td>
 +
                        </tr>
 +
                            <tr>
 +
                            <td align="center" rowspan="2"><font color="#138B3F">ADARYKS(5)</font></td>
 +
                           
 +
                            <td align="center"> <font color="#438CEE"> SILPVTR(2)</font></td>
 +
                            <td align="center" rowspan="2"><font color="#438CEE"> SILPVTR(7)</font> </td>
 +
                           
 +
                        </tr>
 +
                            <tr>
 +
                         
 +
                           
 +
                            <td align="center"> <font color="#F6403A"> MPRLPPA(20)</font></td>
 +
                           
 +
                           
 +
                        </tr>
 +
         </table>
 +
 
 +
            <p>
 +
<b>Table 1B.</b> Table of all peptide sequences observed to bind more than once. The number in parenthesis indicates the number of times the respective peptide was recovered. These candidate peptides were validated for binding affinity with an ELISA.
 +
</p>
 +
</div>     
 +
</div>
 +
    <br>
 +
<p>
 +
 +
We focused our analysis on phage sequences that appeared at least twice. Isolated peptides displayed a range of fabric specificities, sequence motifs and phyicochemical properties (Table 2). Nine peptide sequences were selected for further analysis, chosen with a preference for chemical diversity and highly enriched peptides that appeared in many clones. Five of the nine peptides were fabric-specific, meaning they were isolated from only one fabric, while the others were found to bind multiple fabrics. The peptides were designated FBD 1-9 (Table 3).<br>
 +
   
 +
<h3>Validation of the peptides using ELISA </h3>
 +
    <p>
 +
        The nine selected FBD peptides were validated with ELISA. Briefly, a clonal library of phage expressing each peptide was incubated with each of five fabric samples under conditions similar to the original panning experient. Following extensive washing, the fabric was incubated with an anti-phage primary antibody, then a secondary antibody linked to HRP, the Horse Radish Peroxidaze. The HRP enzyme produces a pigmented product, allowing semi-quantitative measurement of the level of fabric-bound phage.<br><br>
 +
 +
       
 +
 
 +
        In figure 1, we compare the ELISA signal before and after washing for phage displaying each of the FBD peptides against each fabric. In most cases, the phage are retained after washing and produce an ELISA signal significantly above control. Peptides were found to have a range of affinities for different fabrics. Notably wool appeared to be difficult to bind, with only FBD9 staying bound after washing.<br><br>
 +
 +
        Repeated washing reduced signal, allowing a quantitative measurement of binding affinitiy. In a typical case of FBD9 binding to cotton, more than 50% signal was retained after 10 rounds of washing. Analysis of these data is ongoing, with the goal of estimating the KD for each binding interaction.
 +
        </p>
 +
        <div id="figurebox">
 
         <div style="text-align:center;">
 
         <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"/>
+
        <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:500px;">
 +
        <p>
 +
        <b>Figure 1</b> Fabric binding domain validation by ELISA. <b>A.</b> ELISA for each FBD incubated with each fabric after two rounds of washing, normalized to the level after a single round. <b>B.</b> Control matrix repeating the procedure of panel A without phage. <b>C.</b> ELISA signal determined ater multple rounds of washing. Points represent individual measurements from three replicates. Lines represent best-fit to an exponential decay model.
 
         </div>
 
         </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>
 
         </div>
        FBD4-
+
         </p>
         <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>
<br>
+
    <br>
 
+
    <br>
 +
   
 +
   
 
<h2 class="red">Catechol dioxydases were expressed with and without Fabric Binding Domains and their functionality was demonstrated in both cases</h2>
 
<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”>  
 
  <p class=”input”>  

Revision as of 01:41, 20 October 2016


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.
Our phage display experiments were performed with the commercial Ph.D.-7 Phage Display Peptide Library (NEB # E8100S). This library consists 100 copies each of 109 phages expressing a randomized 7-mer peptide fused to the phage N-terminal pIII coat protein. The displayed peptide is separated by a short Gly-Gly-Gly-Ser linker to minimize interactions between the displayed peptide and the phage itself.

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 (Table 1).

COTTON LINEN SILK WOOL POLYESTER
GVLRYAP STNPTSL SILPVTR ADIRHIK MPRLPPA
RLLQYNS MPRLPPA MPRLPPA YMGPSKT MPRLPPH
GVKSEQL MPRLPPA MPRLPPA QFDHWRN MPRLPPA
WHLPAQR MPRLPPA AQSNPKN MRLSVPN MPRLPPA
ELAGTTW MPRLPPA KNANSRE ADARYKS MPRLPPA
MSNTLDP SLLTHNM GKNLMNM SILPVTR ADARYKS
MPRLPPA MPRLPPA MPRLPPA SILPVTR VFQTTYK
ISTTLFP MPRLPPA MPRLPPA ADARYKS ASSHIHH
TTHPRWG MPRLPPA MPRLPPA PSNRQNT GASNIWN
SFLVTRN MPRLPPA KTAMKGP SILPVTR GASNIWN
ASSHIHH MPRLPPA TSNRAPY GSTSFSK ISTTLFP
ALANFEP MPRLPPA ADARYKS ADARYKS GALAKDE
MRLSVPN MPRLPPA MPRLPPA SILPVTR MRLSVPN
MPRLPPA XPRLPPA MPRLPPA ADARYKS MPRLPPA
ERGFLLL QPIYRVQ ADARYKS SILPVTR MPRLPPA
SIHERAK MPRLPPA MPRLPPA QFDHWRN ADARYKS
VECINNC WTNVFVG DETCSSM GQSVVSL ADARYKS
ASSHIHH XPRLPPX MPRLPPA ASSHIHH HWNTVVS
HYPPVDD MPRLPPA MPRLPPA MPRLPPA METVVSS
ASSHIHH XPXXPPT XPRLPPA ASSHIHH MQEMRQM
ADARYKS MPRLPPA MPRLPPA ADARYKS SNYHWRM
TSDATQR XPRLXPA MPRLPPA ADARYKS NNSVSMN
HNWMHQN MPRLPPX FPSPMVG SILPVTR SILPVTR
ADARYKS MPRLPPA AWPYVTL MRLSVPN MPRLPPA
ADARYKS MPRLPPA AQSNPKN HDSPTAA MPRLPPA
TDHAHRY ASPDQEK KNANSRE MPRLPPA FRKKRKS
SVVMPHG MPRLPPA MPRLPPA ADARYKS TESAPTL
ADARYKS QFPPPPG MPRLPPX ADIRHIK SLETMSN
FSRSNNT HDSPTAA MPRLPPX YMGPSKT MPRLPPA
TDMTAPK MPRLPPA HDVMWQR SILPVTR MPRLPPA
MTQQLHT MPRLPPA MPRLPPA ASSHIHH MPRLPPA
SSHSVQR TNLHINP HWNTVVS TVHVHKT VPRLPPA
GLHYDHS AGHVVPR MLQGNGY ADIRHIK MPRLPPA
ASSHIHH HWNTVVS ASSHIHH YMGPSKT MPRLPPA
DPRLSPT SILPVTR SILPVTR GSTSFSK MPRLPPA
QGDYFTY TLINYRG AGHVVPR SILPVTR MPRLPPA
VTLPDPR ADARYKS MPRLPPA TRPTDTI MPRLPPA
VTLPDPR MPRLPPA MPRXPPA GSTSFSK MPRLPPA
FSRSNNT SILPVXR MPRLPPA HDSPTAA
ADARYKS HDVMWQR MPRLPPA YMGPSKT
SILPVTR

Table 1A. Novel fabric binding domains with diverse properties.


COTTON LINEN SILK WOOL POLYESTER
FSRSNNT(2) SILPVTR(2) AQSNPKN(2) MRLSVPN(2) ADARYKS(3)
MPRLPPA(2) AWPYVTL(2) ASSHIHH(3)
ASSHIHH(4) MPRLPPA(25) KNANSRE(2) ADARYKS(6) MPRLPPA(20)
ADARYKS(5) SILPVTR(2) SILPVTR(7)
MPRLPPA(20)

Table 1B. Table of all peptide sequences observed to bind more than once. The number in parenthesis indicates the number of times the respective peptide was recovered. These candidate peptides were validated for binding affinity with an ELISA.


We focused our analysis on phage sequences that appeared at least twice. Isolated peptides displayed a range of fabric specificities, sequence motifs and phyicochemical properties (Table 2). Nine peptide sequences were selected for further analysis, chosen with a preference for chemical diversity and highly enriched peptides that appeared in many clones. Five of the nine peptides were fabric-specific, meaning they were isolated from only one fabric, while the others were found to bind multiple fabrics. The peptides were designated FBD 1-9 (Table 3).

Validation of the peptides using ELISA

The nine selected FBD peptides were validated with ELISA. Briefly, a clonal library of phage expressing each peptide was incubated with each of five fabric samples under conditions similar to the original panning experient. Following extensive washing, the fabric was incubated with an anti-phage primary antibody, then a secondary antibody linked to HRP, the Horse Radish Peroxidaze. The HRP enzyme produces a pigmented product, allowing semi-quantitative measurement of the level of fabric-bound phage.

In figure 1, we compare the ELISA signal before and after washing for phage displaying each of the FBD peptides against each fabric. In most cases, the phage are retained after washing and produce an ELISA signal significantly above control. Peptides were found to have a range of affinities for different fabrics. Notably wool appeared to be difficult to bind, with only FBD9 staying bound after washing.

Repeated washing reduced signal, allowing a quantitative measurement of binding affinitiy. In a typical case of FBD9 binding to cotton, more than 50% signal was retained after 10 rounds of washing. Analysis of these data is ongoing, with the goal of estimating the KD for each binding interaction.

Paris_bettencourt-Selected_ELISA_Validation

Figure 1 Fabric binding domain validation by ELISA. A. ELISA for each FBD incubated with each fabric after two rounds of washing, normalized to the level after a single round. B. Control matrix repeating the procedure of panel A without phage. C. ELISA signal determined ater multple rounds of washing. Points represent individual measurements from three replicates. Lines represent best-fit to an exponential decay model.




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

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



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