Difference between revisions of "Team:DTU-Denmark/Project/Detection"

 
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  >Home
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                  >Home
                                          </a></li>
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                                          </a></li>
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<a href="/Team:DTU-Denmark/Attributions"
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                <a href="/Team:DTU-Denmark/Attributions"
  >Attributions
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                  >Attributions
                                          </a></li></ul></li>
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                >
 +
                <a href=""
 +
                  class="dropdown-toggle"
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                  data-toggle="dropdown"
 +
                  role="button"
 +
                  aria-expanded="false">Project
 +
                  <span class="caret"></span></a><ul class="dropdown-menu" role="menu">
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Overview"
 +
                  >Overview
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Background"
 +
                  >Background
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/MAGE"
 +
                  >MAGE subtilis
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Tyrocidine"
 +
                  >Tyrocidine
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Chip"
 +
                  >Lab-on-a-disc
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Inteins"
 +
                  >Inteins
 +
                  </a></li>
 +
                <li class="active">
 +
                <a href="/Team:DTU-Denmark/Project/Detection"
 +
                  >Detection of NRP
 +
                  </a></li></ul></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Practices"
 +
                  >Human Practices
 +
                  </a></li>
 +
                <li
 +
               
 
                      
 
                      
                 
+
               
  >
+
<a href="/Team:DTU-Denmark"
+
  class="dropdown-toggle"
+
  data-toggle="dropdown"
+
  role="button"
+
  aria-expanded="false">Project
+
                                          <span class="caret"></span></a><ul class="dropdown-menu" role="menu">
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Overview"
+
  >Overview
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Background"
+
  >Background
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/MAGE"
+
  >MAGE Subtilis
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Surfactin"
+
  >Surfactin
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Tyrocidine"
+
  >Tyrocidine
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Chip"
+
  >Lab-on-a-disc
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/Intein"
+
  >Intein
+
                                          </a></li>
+
<li class="active">
+
<a href="/Team:DTU-Denmark/Project/Detection"
+
  >Detection of NRP
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Project/POC_MAGE"
+
  >Proof of concept of MAGE in B. subtilis
+
                                          </a></li></ul></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Practices"
+
  >Human Practices
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Part_Collection"
+
  >Parts
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Journal"
+
  >Journal
+
                                          </a></li>
+
<li >
+
<a href="/Team:DTU-Denmark/Software"
+
  >Software
+
                                          </a></li>
+
<li
+
+
 
                      
 
                      
                 
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 +
                >
 +
                <a href=""
 +
                  class="dropdown-toggle"
 +
                  data-toggle="dropdown"
 +
                  role="button"
 +
                  aria-expanded="false">Parts Collection
 +
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 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Parts"
 +
                  >Parts
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Description"
 +
                  >Characterisation of xylR
 +
                  </a></li></ul></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Journal"
 +
                  >Journal
 +
                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Software"
 +
                  >Software
 +
                  </a></li>
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<a href="/Team:DTU-Denmark"
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  class="dropdown-toggle"
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                >
  data-toggle="dropdown"
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  aria-expanded="false">Achievements
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                  aria-expanded="false">Achievements
<a href="/Team:DTU-Denmark/Achievements"
+
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  >Overview of Achievements
+
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                                          </a></li>
+
                <a href="/Team:DTU-Denmark/Achievements"
<li >
+
                  >Key Achievements
<a href="/Team:DTU-Denmark/Collaborations"
+
                  </a></li>
  >Collaborations
+
                <li >
                                          </a></li>
+
                <a href="/Team:DTU-Denmark/Collaborations"
<li >
+
                  >Collaborations
<a href="/Team:DTU-Denmark/Judging%20Form"
+
                  </a></li>
  >Judging Form
+
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                                          </a></li></ul></li>
+
                <a href="/Team:DTU-Denmark/Judging_Form"
 +
                  >Judging Form
 +
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          <li><a class="page-scroll" href="#Purpose">Purpose</a></li><li><a class="page-scroll" href="#Achivements">Achivements</a></li><li><a class="page-scroll" href="#Background">Background</a></li><li><a class="page-scroll" href="#Method">Method</a></li><li><a class="page-scroll" href="#Results">Results</a></li><li><a class="page-scroll" href="#Discussion">Discussion</a></li><li><a class="page-scroll" href="#References">References</a></li>
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      <li><a class="page-scroll" href="#Purpose">Purpose</a></li><li><a class="page-scroll" href="#Achievements">Achievements</a></li><li><a class="page-scroll" href="#Background">Background</a></li><li><a class="page-scroll" href="#ExperimentalWork">Experimental Work</a></li><li><a class="page-scroll" href="#Results">Results</a></li><li><a class="page-scroll" href="#Discussion">Discussion</a></li><li><a class="page-scroll" href="#References">References</a></li>
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           <div class="col-md-12">
 
           <div class="col-md-12">
 
              
 
              
            <h3><br></h3>
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<h3><br></h3>
            <h1>Detection of NRP</h1>
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<h1>Detection of NRP</h1>
           
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             <hr/>
 
             <hr/>
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             <ul class="list-inline hidden-xs hidden-sm">
 
                
 
                
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               <li>
 
               <li>
                 <a id="Achivements-submenu" class="btn btn-default btn-transparent btn-lg page-scroll" href="#Achivements">Achivements</a>
+
                 <a id="Achievements-submenu" class="btn btn-default btn-transparent btn-lg page-scroll" href="#Achievements">Achievements</a>
 
               </li>
 
               </li>
 
                
 
                
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               <li>
 
               <li>
                 <a id="Method-submenu" class="btn btn-default btn-transparent btn-lg page-scroll" href="#Method">Method</a>
+
                 <a id="ExperimentalWork-submenu" class="btn btn-default btn-transparent btn-lg page-scroll" href="#ExperimentalWork">Experimental Work</a>
 
               </li>
 
               </li>
 
                
 
                
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               <li>
 
               <li>
                 <a id="Achivements-submenu" class="page-scroll" href="#Achivements">Achivements</a>
+
                 <a id="Achievements-submenu" class="page-scroll" href="#Achievements">Achievements</a>
 
               </li>
 
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     </div>
 
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<div id="Purpose">
 
<div id="Purpose">
 
   <div class="container">
 
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 +
 
 
     <div class="row col-md-12">
 
     <div class="row col-md-12">
 
       <h1>
 
       <h1>
 
         Purpose
 
         Purpose
 
       </h1>
 
       </h1>
      <p>Separation and identification of our non-ribosomal peptide synthetase (NRPS) products was determined by ultra-high performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS). NRPS variants were identified by changes in mass/z and column retention time.&nbsp;Development of a matrix assisted laser desorption ionization time-of-flight mass spec (MALDI-TOF-MS) method will allow for future rapid analysis of oligo-recombineered strains producing a library of NRPS antibiotic products. DTU&rsquo;s 2015 iGEM team was able to procure the assistance of the DTU Metabolomics Platform group, which specializes in analytical chromatography and is recognized in our <a href="/Team:DTU-Denmark/Team#Attributions">attributions page</a>. With assistance from our advisor, a protocol was established to lyse the cells and extract the tyrocidine and surfactin molecules before they were run on the liquid chromatography mass spec (LCMS) for analysis.</p>
+
       
 +
        <p style="text-align: justify;">Separation and identification of our non-ribosomal peptide synthetase (NRPS) products, was determined by ultra-high performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS). NRPS variants were identified by changes in mass/z and column retention time.&nbsp;<br />
 +
Development of a matrix assisted laser desorption ionization time-of-flight mass spec (MALDI-TOF-MS) method,&nbsp;will allow for a more rapid future analysis of oligo-recombineered strains, producing a library of NRPS antibiotic products. DTU&rsquo;s 2015 iGEM team was able to, with the assistance of the DTU Metabolomics Platform group, which specializes in analytical chromatography&nbsp;(and is recognized in our <a href="/Team:DTU-Denmark/Team#Attributions">attributions page</a>), and&nbsp;with the assistance from our advisor, to produce a protocol for&nbsp;lysing of&nbsp;the cells, and extraction of the tyrocidine and surfactin molecules,&nbsp;before they were run on the liquid chromatography mass spec (LCMS) for analysis.</p>
  
 +
       
 
     </div>
 
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   </div>
 
</div>
 
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<div id="Achivements">
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<div id="Achievements">
 
   <div class="container">
 
   <div class="container">
 +
 
 
     <div class="row col-md-12">
 
     <div class="row col-md-12">
 
       <h1>
 
       <h1>
         Achivements
+
         Achievements
 
       </h1>
 
       </h1>
     
+
       
 +
        <ul>
 +
<li>Extraction protocol developed for Tyrocidine A-E</li>
 +
<li>Extraction protocol developed for Surfactin</li>
 +
<li>HPLC detection of Tyrocidine A-E in Brevibacillus parabrevis compared to analytical standard (Tyrothricin)</li>
 +
<li>HPLC detection of Surfactin in Bacillus subtilis</li>
 +
<li>Detection of Tyrocidine A and Surfactin in MALDI-TOF</li>
 +
<li>Detection of MAGE modified Surfactin variant</li>
 +
</ul>
 +
 
 +
       
 
     </div>
 
     </div>
 +
   
 
   </div>
 
   </div>
 
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<div id="Background">
 
   <div class="container">
 
   <div class="container">
 +
 
 
     <div class="row col-md-12">
 
     <div class="row col-md-12">
 
       <h1>
 
       <h1>
 
         Background
 
         Background
 
       </h1>
 
       </h1>
      <p>Tyrocidine and surfactin can both be separated by liquid chromatography, which separates molecules in a liquid mobile phase as they pass over a solid stationary media in a column. Molecules bind to or elute off the column based on their differing physical properties; including electronegativity, polarity, hydrophobicity, or size. Different detection methods (eg. mass spectroscopy, fluorescence detection, UV/Visible light spectroscopy) can then be coupled to the HPLC to analyze the compounds that were separated from the original solution. The reversed phase liquid chromatography column used in our method has a non-polar stationary phase and polar mobile phase, which causes polar molecules to elute before non-polar compounds. Initial preparative steps remove large contaminants from the sample such as the cell membranes or organelles, which can clog and damage the columns.</p>
+
       
 +
        <p style="text-align: justify;">Tyrocidine and surfactin can both be separated by liquid chromatography, which separates molecules in a liquid mobile phase, as they pass over a solid stationary media in a column. Molecules bind to or elute off the column based on their differing physical properties; including electronegativity, polarity, hydrophobicity, or size. Different detection methods (eg. mass spectroscopy, fluorescence detection, UV/Visible light spectroscopy) can then be coupled to the HPLC, to analyze the compounds that were separated from the original solution. The reversed phase liquid chromatography column, used in our method has a non-polar stationary phase, and polar mobile phase, which causes polar molecules to elute before non-polar compounds. Initial preparative steps removed large contaminants from the sample, such as the cell membranes or organelles, which can clog and damage the columns.</p>
  
<p>As there are many different metabolites produced by prokaryotic and eukaryotic cells, it is important to have an idea of the chemical properties and mass of the target compound. Tyrocidine is an amphiphilic, cyclic peptide naturally produced by an NRPS&nbsp;in <em>Bacillus brevis </em>with antimicrobial properties against gram-positive bacteria [1]. It functions by&nbsp;disrupting cell membranes, which explains its broad efficacy, but it&nbsp;has been shown to have hemolytic activity [2], making it unsuitable for intravenous use. Tyrocidine has been reported in the literature to have 5 main forms with the general peptide sequence of &hellip;D-Phe<sub>1</sub>/Tyr<sub>1</sub> &ndash; Pro<sub>2</sub> &ndash; Phe<sub>3</sub>/Trp<sub>3</sub>/Tyr<sub>3</sub> &ndash; D-Phe<sub>4</sub>/D-Trp<sub>4</sub>/D-Tyr<sub>4&shy;</sub> &ndash; Asn<sub>5</sub> &ndash; Gln<sub>6</sub>/Asp<sub>6</sub> &ndash; Val<sub>8</sub> &ndash; Orn<sub>9</sub> &ndash; Leu<sub>10</sub>&hellip; [3]</p>
+
<p style="text-align: justify;">As there are many different metabolites produced by prokaryotic and eukaryotic cells, it is important to have an idea of the chemical properties and mass of the target compound. Tyrocidine is an amphiphilic, cyclic peptide naturally produced by a non-ribosomal peptide synthetase (NRPS) in <em>Bacillus brevis, </em>with antimicrobial properties against gram-positive bacteria [1]. It functions by&nbsp;disrupting cell membranes, which explains its broad efficacy, but it&nbsp;has been shown to have hemolytic activity [2], making it unsuitable for intravenous use. Tyrocidine has been reported in the literature to have 5 main forms with the general peptide sequence of &hellip;D-Phe<sub>1</sub>/Tyr<sub>1</sub> &ndash; Pro<sub>2</sub> &ndash; Phe<sub>3</sub>/Trp<sub>3</sub>/Tyr<sub>3</sub> &ndash; D-Phe<sub>4</sub>/D-Trp<sub>4</sub>/D-Tyr<sub>4&shy;</sub> &ndash; Asn<sub>5</sub> &ndash; Gln<sub>6</sub>/Asp<sub>6</sub> &ndash; Val<sub>8</sub> &ndash; Orn<sub>9</sub> &ndash; Leu<sub>10</sub>&hellip; [3]</p>
  
<p>&nbsp;</p>
+
<table border="0" cellpadding="0" cellspacing="0" class="table table-hover">
 +
<thead>
 +
<tr>
 +
<th scope="col" style="width: 175px;">
 +
<p style="text-align: justify;">NRPS Product</p>
 +
</th>
 +
<th scope="col" style="width: 172px;">
 +
<p style="text-align: justify;">Amino Acid Sequence</p>
 +
</th>
 +
<th scope="col" style="width: 138px;">
 +
<p style="text-align: justify;">Molecular Formula</p>
 +
</th>
 +
<th scope="col" style="width: 138px;">
 +
<p style="text-align: justify;">MW, g/mole</p>
 +
</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Tyrocidine A</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">F<sub>1</sub>P<sub>2</sub>F<sub>3</sub>F<sub>4</sub>N<sub>5</sub>Q<sub>6</sub>Y<sub>7</sub>V<sub>8</sub>O<sub>9</sub>L<sub>10</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C66H87N13O13</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1270.6619</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Tyrocidine B</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">F<sub>1</sub>P<sub>2</sub>W<sub>3</sub>F<sub>4</sub>N<sub>5</sub>Q<sub>6</sub>Y<sub>7</sub>V<sub>8</sub>O<sub>9</sub>L<sub>10</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C68H88N14O13</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1309.6728</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Tyrocidine C</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">F<sub>1</sub>P<sub>2</sub>W<sub>3</sub>W<sub>4</sub>N<sub>5</sub>Q<sub>6</sub>Y<sub>7</sub>V<sub>8</sub>O<sub>9</sub>L<sub>10</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C70H89N15O13</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1348.6837</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Tyrocidine D</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">F<sub>1</sub>P<sub>2</sub>W<sub>3</sub>W<sub>4</sub>N<sub>5</sub>Q<sub>6</sub>F<sub>7</sub>V<sub>8</sub>O<sub>9</sub>L<sub>10</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C72H90N16O12</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1371.6997</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Tyrocidine E</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">F<sub>1</sub>P<sub>2</sub>F<sub>3</sub>F<sub>4</sub>N<sub>5</sub>Q<sub>6</sub>F<sub>7</sub>V<sub>8</sub>O<sub>9</sub>L<sub>10</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C66H87N13O12</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1254.6670</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Surfactin</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">Q<sub>1</sub>L<sub>2</sub>L<sub>3</sub>V<sub>4</sub>D<sub>5</sub>L<sub>6</sub>L<sub>7</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C53H93N7O13</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1036.34</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:175px;">
 +
<p style="text-align: justify;">Surfactin Variant</p>
 +
</td>
 +
<td style="width:172px;">
 +
<p style="text-align: justify;">Q<sub>1</sub>L<sub>2</sub>L<sub>3</sub>V<sub>4</sub>N<sub>5</sub>L<sub>6</sub>L<sub>7</sub></p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">C53H94N8O12</p>
 +
</td>
 +
<td style="width:138px;">
 +
<p style="text-align: justify;">1035.36</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
  
<p><img alt="" src="/wiki/images/7/78/DTU-Denmark_surfactin_highlight.png" style="height: 214px; width: 300px;" /></p>
+
<p style="text-align: justify;">Surfactin has the formula C<sub>53</sub>H<sub>93</sub>N<sub>7</sub>O<sub>13</sub> and is composed of a 7 amino acid cylic peptide with a hydrophobic fatty acid chain (Glu<sub>1</sub> &ndash; Leu<sub>2</sub> &ndash; D-Leu<sub>3&shy;&shy;</sub> &ndash; Val<sub>4</sub> &ndash; Asp<sub>5</sub> &ndash; D-Leu<sub>6</sub> &ndash; Leu<sub>7</sub> &ndash; &beta;-OH-C<sub>13-15</sub>) [4]. It is natively produced by <em>Bacillus subtilis</em> NRPS and in addition to functioning as an antibiotic it has detergent &ndash;like surfactant properties, antiviral and antifungal activities, and the capability to lyse red blood cells [5] making it another interesting candidate for a&nbsp;oligo-recombineering library creation.</p>
  
<p>Figure 1: Surfactin, highlighted with the blue circle at the location of the mutation from an aspartic acid residue to asparagine</p>
+
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/0/07/DTU-Denmark_tyrocidine_A.png" style="border-width: 1px; border-style: solid; width: 512px; height: 492px;" /></p>
  
<p><img alt="" src="/wiki/images/0/07/DTU-Denmark_tyrocidine_A.png" style="height: 384px; width: 300px;" /></p>
+
<p style="text-align: justify;">Figure 1: cyclic peptide,&nbsp;Tyrocidine A&nbsp;</p>
  
<p>Figure 2: cyclic peptide,&nbsp;Tyrocidine A</p>
+
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/7/78/DTU-Denmark_surfactin_highlight.png" style="border-width: 1px; border-style: solid; width: 512px; height: 365px;" /></p>
  
 +
<p style="text-align: justify;">Figure 2:&nbsp;Surfactin, highlighted with the blue circle at the location of the mutation from an aspartic acid residue to asparagine</p>
 +
 +
       
 
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 +
   
 
   </div>
 
   </div>
 
</div>
 
</div>
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+
       
<div id="Method">
+
<div id="ExperimentalWork">
 
   <div class="container">
 
   <div class="container">
 +
 
 
     <div class="row col-md-12">
 
     <div class="row col-md-12">
 
       <h1>
 
       <h1>
         Method
+
         Experimental Work
 
       </h1>
 
       </h1>
      <p>&nbsp;</p>
+
       
 +
        <p style="text-align: justify;">Tyrocidine was first detected in our native <i>DMZ362 Brevibacillus parabrevis</i> strain using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Initial experiments were done to develop an extraction method for the compound of interest using the <i>DMZ362 Brevibacillus parabrevis</i> as a positive control, <i>Bacillus subtilis</i> as a negative control, and an analytical standard, Tyrothricin, which contains tyrocidine A-E purified from <i>Bacillus brevis</i>. After testing for the compound in both the agar media and biomass, an extraction protocol was developed for use with future tyrocidine antibiotic libraries.</p>
  
 
<div aria-multiselectable="true" class="panel-group" id="accordion" role="tablist">
 
<div aria-multiselectable="true" class="panel-group" id="accordion" role="tablist">
 
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<div class="panel panel-default">
 
<div class="panel-heading" id="headingOne" role="tab">
 
<div class="panel-heading" id="headingOne" role="tab">
<h4 class="panel-title"><a aria-controls="collapseOne" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseOne" role="button">HPLC Sample Preparation: Tyrocidine Extraction</a></h4>
+
<h4 class="panel-title" style="text-align: justify;"><a aria-controls="collapseOne" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseOne" role="button">HPLC Sample Preparation: Tyrocidine Extraction</a></h4>
 
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<ol>
 
<ol>
<li>Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.</li>
+
<li style="text-align: justify;">Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.</li>
<li>5 mL of methanol was added to the biomass to lyse the cell membranes before they were centrifuged at 4700 x g for 5 minutes to pellet the cell material.</li>
+
<li style="text-align: justify;">5 mL of methanol was added to the biomass to lyse the cell membranes before they were centrifuged at 4700 x g for 5 minutes to pellet the cell material.</li>
<li>Supernatant was carefully removed by pipetting 250 &mu;L into an HPLC vial for analysis</li>
+
<li style="text-align: justify;">Supernatant was carefully removed by pipetting 250 &mu;L into an HPLC vial for analysis</li>
 
</ol>
 
</ol>
 
</div>
 
</div>
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<div class="panel panel-default">
 
<div class="panel-heading" id="headingTwo" role="tab">
 
<div class="panel-heading" id="headingTwo" role="tab">
<h4 class="panel-title"><a aria-controls="collapseTwo" aria-expanded="false" class="collapsed" data-parent="#accordion2" data-toggle="collapse" href="#collapseTwo" role="button">HPLC Sample Preparation: Surfactin Extraction</a></h4>
+
<h4 class="panel-title" style="text-align: justify;"><a aria-controls="collapseTwo" aria-expanded="false" class="collapsed" data-parent="#accordion2" data-toggle="collapse" href="#collapseTwo" role="button">HPLC Sample Preparation: Surfactin Extraction</a></h4>
 
</div>
 
</div>
  
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<div class="panel-body">
 
<div class="panel-body">
 
<ol>
 
<ol>
<li>Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.</li>
+
<li style="text-align: justify;">Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.</li>
<li>5 mL of ethyl acetate with 1% formic acid was added to the sample to ensure positively charged residues were protonated and the surfactin was extracted from the cell. Sample was sonicated for 5 minutes before it was centrifuged at 4700 x g for 5 minutes to pellet the cell material.</li>
+
<li style="text-align: justify;">5 mL of ethyl acetate with 1% formic acid was added to the sample to ensure positively charged residues were protonated and the surfactin was extracted from the cell. Sample was sonicated for 5 minutes before it was centrifuged at 4700 x g for 5 minutes to pellet the cell material.</li>
<li>Supernatant was carefully removed by pipetting into a fresh 15 mL Falcon tube.</li>
+
<li style="text-align: justify;">Supernatant was carefully removed by pipetting into a fresh 15 mL Falcon tube.</li>
<li>Ethyl acetate was evaporated with gentle N<sub>2</sub> gas flow in a fume hood and the extract was re-dissolved in 300 &mu;L of methanol.</li>
+
<li style="text-align: justify;">Ethyl acetate was evaporated with gentle N<sub>2</sub> gas flow in a fume hood and the extract was re-dissolved in 300 &mu;L of methanol.</li>
<li>Methanol soluble extract was vortexed and centrifuged at 4700 x g for 5 minutes and 250 &mu;L of sample was pipetted into an HPLC vial for analysis.</li>
+
<li style="text-align: justify;">Methanol soluble extract was vortexed and centrifuged at 4700 x g for 5 minutes and 250 &mu;L of sample was pipetted into an HPLC vial for analysis.</li>
 
</ol>
 
</ol>
 
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion3" data-toggle="collapse" href="#collapseThree" role="button">UHPLC-DAD-QTOFMS Method</a></h4>
+
<h4 class="panel-title" style="text-align: justify;"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion3" data-toggle="collapse" href="#collapseThree" role="button">UHPLC-DAD-QTOFMS Method</a></h4>
 
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<div aria-labelledby="headingThree" class="panel-collapse collapse" id="collapseThree" role="tabpanel">
<div class="panel-body">Extracted samples (in methanol) were separated by ultra-high performance liquid chromatography over an Agilent Poroshell 120 phenyl-hexyl column (2.1 x 250 mm, 2.7 &mu;m). The detection was measured with a diode array detection coupled to quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS). Samples were run with a &nbsp;a linear gradient from 10 to&nbsp; 100% solvent B in 15 min, followed by 100% B for 2 min and rapidly returned to 10% B to equilibrate for 3 minutes at a flow rate of 0.35 mL/min (60&deg;C). Solvent A was Milli-Q Water, solvent B was acetonitrile and both were buffered in 20 mM formic acid. The injection volume was 1 &mu;L. MS detection was measured on an Agilent 6545 QTOF MS with an electrospray ion source. Capillary voltage was set to 4000 V with a nozzle voltage of 500 V. Mass spectra were recorded for m/z 85&ndash;1700 in MS mode and m/z 30&ndash;1700 in MS/MS mode, with an acquisition rate of 10 spectra/s. Analysis of the spectra was done using Agilent MassHunter version B.07.00 to determine the presecnce of tyrocidine, surfactin, and modified surfactin using the &ldquo;find by formula&rdquo; function, with the formulas found in the table above. Manual inspection of the extracted ion chromatogram traces for [surfactin+H]<sup>+</sup> at m/z of 1035.706 and [surfactin + Na]<sup>+</sup> at m/z of was also performed.</div>
+
<div class="panel-body" style="text-align: justify;">Extracted samples (in methanol) were separated by ultra-high performance liquid chromatography over an Agilent Poroshell 120 phenyl-hexyl column (2.1 x 250 mm, 2.7 &mu;m). The detection was measured with a diode array detection coupled to quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS). Samples were run with a &nbsp;a linear gradient from 10 to&nbsp; 100% solvent B in 15 min, followed by 100% B for 2 min and rapidly returned to 10% B to equilibrate for 3 minutes at a flow rate of 0.35 mL/min (60&deg;C). Solvent A was Milli-Q Water, solvent B was acetonitrile and both were buffered in 20 mM formic acid. The injection volume was 1 &mu;L. MS detection was measured on an Agilent 6545 QTOF MS with an electrospray ion source. Capillary voltage was set to 4000 V with a nozzle voltage of 500 V. Mass spectra were recorded for m/z 85&ndash;1700 in MS mode and m/z 30&ndash;1700 in MS/MS mode, with an acquisition rate of 10 spectra/s. Analysis of the spectra was done using Agilent MassHunter version B.07.00 to determine the presecnce of tyrocidine, surfactin, and modified surfactin using the &ldquo;find by formula&rdquo; function, with the formulas found in the table above. Manual inspection of the extracted ion chromatogram traces for [surfactin+H]<sup>+</sup> at m/z of 1035.706 and [surfactin + Na]<sup>+</sup> at m/z of was also performed.</div>
 
</div>
 
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</div>
 
</div>
  
<p>&nbsp;</p>
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<div class="panel-heading" id="headingFour" role="tab">
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<h4 class="panel-title" style="text-align: justify;"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion3" data-toggle="collapse" href="#collapseFour" role="button">MALDI-TOF-MS Equipment and Method</a></h4>
 +
</div>
  
 +
<div aria-labelledby="headingThree" class="panel-collapse collapse" id="collapseFour" role="tabpanel">
 +
<div class="panel-body" style="text-align: justify;">Mass spectra were determined using a Bruker Autoflex II MALDI-TOF instrument. Extracted samples were spotted on an AnchorChip MALDI target plate (1 uL) and allowed to dry before being covered (1 uL) with Universal Matrix from Sigma-Aldrich (1:1 ratio of 2,5-Dihydroxybenzoic Acid to alpha-Cyano-4-hydroxycinnamic acid). The machine was operated in both linear and reflectron mode with a mass range of 500-3000. Positive and negative ion detection and reflector mode were used. Analysis of extracted samples in methanol (previously verified by HPLC-MS) by FlexAnalysis Version 3.4 indicate that tyrocidine and surfactin was clearly detectable. Surfactin was detectable in negative mode with reduced background noise compared to the positive mode, making verification a simple task.</div>
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<div id="Results">
 
<div id="Results">
 
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       <h1>
 
       <h1>
 
         Results
 
         Results
 
       </h1>
 
       </h1>
     
+
       
 +
        <p style="text-align: justify;">Tyrocidine HPLC-MS:</p>
 +
 
 +
<p style="text-align: justify;">Looking at the extrapolated ion chromatogram for a mass range between 950 - 1300 Tyrocidine is clearly visible in the UHPLC-DAD-QTOFMS running under the described parameters.</p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/5/59/DTU-Denmark_TyroBreviHPLC.png" style="width: 1580px; height: 507px;" /></p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/0/05/DTU-Denmark_TyroBreviandstdHPLC.png" /></p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/b/bc/DTU-Denmark_TyroBrevivsBacHPLC.png" style="width: 1518px; height: 600px;" /></p>
 +
 
 +
<p style="text-align: justify;">Surfactin MALDI-TOF:</p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/7/72/DTU-Denmark_SurMALDIRefNeg.png" style="width: 1476px; height: 953px;" /></p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/e/e1/DTU-Denmark_SurMaldiLinPos.png" /></p>
 +
 
 +
<p style="text-align: justify;">Surfactin HPLC-MS:</p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/0/0f/DTU-Denmark_HPLCSurMut.png" /></p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/b/bf/DTU-Denmark_HPLCSurOver.png" style="width: 1505px; height: 514px;" /></p>
 +
 
 +
<p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/5/5f/DTU-Denmark_HPLCSurESI.png" style="width: 1678px; height: 364px;" /></p>
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         Discussion
 
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       </h1>
     
+
       
 +
        <p style="text-align: justify;">Surfactin and Tyrosidine were detectable with MALDI-TOF&nbsp;after optimizing their extraction methods. Tyrocidine is not shown here, but was detectable using the positive scan mode with the reflectron. See below for detection of the exact mass/z peaks of surfactin spotted on the MALDI plates. Surfactin was detectable using the negative scan mode making for easier identification due to the lack of other compounds ionizing with that detection method.</p>
 +
 
 +
<p style="text-align: justify;">Freshly prepared colonies that were recombineered with the previously described MAGE method to create an NRPS that could switch out the aspartic acid for asparagine, were extracted after only 12 hours of growth, and analyzed on the UHPLC-DAD-QTOFMS, showed very small amounts of detectable surfactin. This result is expected as surfactin is produced during sporulation and this occurs with older bacterial samples. In addition only about 4% of the transformants were expected to have the mutation. One sample was run that showed a mass spec correlating to the expected mass of the mutant. This result indicates that the MAGE method was successful! More samples will be submitted for verification of this result with older streaked B. subtilis mutants biomass.</p>
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       <h1>
 
       <h1>
 
         References
 
         References
 
       </h1>
 
       </h1>
      <ol>
 
 
          
 
          
          <li>Kohli, R. M., Walsh, C. T., & Burkart, M. D. (2002). Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature, 418(6898), 658–661. <a href="http://dx.doi.org/10.1038/nature00907" target="_blank">doi:10.1038/nature00907</a></li>
+
<ol>
       
+
   
          <li>Qin, C., Bu, X., Wu, X., & Guo, Z. (2003). A Chemical Approach to Generate Molecular Diversity Based on the Scaffold of Cyclic Decapeptide Antibiotic Tyrocidine A. Journal of Combinatorial Chemistry, 5(4), 353–355. <a href="http://dx.doi.org/10.1021/cc0300255" target="_blank">doi:10.1021/cc0300255</a></li>
+
  <li>Kohli, R. M., Walsh, C. T., & Burkart, M. D. (2002). Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature, 418(6898), 658–661. <a href="http://dx.doi.org/10.1038/nature00907" target="_blank">doi:10.1038/nature00907</a></li>
       
+
   
          <li>Hitzeroth, G., Vater, J., Franke, P., Gebhardt, K., & Fiedler, H.-P. (2005). Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry andin situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides. Rapid Commun. Mass Spectrom., 19(20), 2935–2942. <a href="http://dx.doi.org/10.1002/rcm.2155" target="_blank">doi:10.1002/rcm.2155</a></li>
+
  <li>Qin, C., Bu, X., Wu, X., & Guo, Z. (2003). A Chemical Approach to Generate Molecular Diversity Based on the Scaffold of Cyclic Decapeptide Antibiotic Tyrocidine A. Journal of Combinatorial Chemistry, 5(4), 353–355. <a href="http://dx.doi.org/10.1021/cc0300255" target="_blank">doi:10.1021/cc0300255</a></li>
       
+
   
      </ol>
+
  <li>Hitzeroth, G., Vater, J., Franke, P., Gebhardt, K., & Fiedler, H.-P. (2005). Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry andin situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides. Rapid Commun. Mass Spectrom., 19(20), 2935–2942. <a href="http://dx.doi.org/10.1002/rcm.2155" target="_blank">doi:10.1002/rcm.2155</a></li>
 +
   
 +
  <li>Baumgart, F., Kluge, B., Ullrich, C., Vater, J., &amp; Ziessow, D. (1991). Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy. Biochemical and Biophysical Research Communications, 177(3), 998–1005. &lt;a href=&#34;http://dx.doi.org/10.1016/0006-291x(91)90637-m&#34; target=&#34;_blank&#34;&gt;doi:10.1016/0006-291x(91)90637-m&lt;/a&gt;</li>
 +
   
 +
  <li>Singh, P., & Cameotra, S. S. (2004). Potential applications of microbial surfactants in biomedical sciences. Trends in Biotechnology, 22(3), 142–146. <a href="http://dx.doi.org/10.1016/j.tibtech.2004.01.010" target="_blank">doi:10.1016/j.tibtech.2004.01.010</a></li>
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Latest revision as of 15:14, 9 November 2015

Purpose

Separation and identification of our non-ribosomal peptide synthetase (NRPS) products, was determined by ultra-high performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS). NRPS variants were identified by changes in mass/z and column retention time. 
Development of a matrix assisted laser desorption ionization time-of-flight mass spec (MALDI-TOF-MS) method, will allow for a more rapid future analysis of oligo-recombineered strains, producing a library of NRPS antibiotic products. DTU’s 2015 iGEM team was able to, with the assistance of the DTU Metabolomics Platform group, which specializes in analytical chromatography (and is recognized in our attributions page), and with the assistance from our advisor, to produce a protocol for lysing of the cells, and extraction of the tyrocidine and surfactin molecules, before they were run on the liquid chromatography mass spec (LCMS) for analysis.

Achievements

  • Extraction protocol developed for Tyrocidine A-E
  • Extraction protocol developed for Surfactin
  • HPLC detection of Tyrocidine A-E in Brevibacillus parabrevis compared to analytical standard (Tyrothricin)
  • HPLC detection of Surfactin in Bacillus subtilis
  • Detection of Tyrocidine A and Surfactin in MALDI-TOF
  • Detection of MAGE modified Surfactin variant

Background

Tyrocidine and surfactin can both be separated by liquid chromatography, which separates molecules in a liquid mobile phase, as they pass over a solid stationary media in a column. Molecules bind to or elute off the column based on their differing physical properties; including electronegativity, polarity, hydrophobicity, or size. Different detection methods (eg. mass spectroscopy, fluorescence detection, UV/Visible light spectroscopy) can then be coupled to the HPLC, to analyze the compounds that were separated from the original solution. The reversed phase liquid chromatography column, used in our method has a non-polar stationary phase, and polar mobile phase, which causes polar molecules to elute before non-polar compounds. Initial preparative steps removed large contaminants from the sample, such as the cell membranes or organelles, which can clog and damage the columns.

As there are many different metabolites produced by prokaryotic and eukaryotic cells, it is important to have an idea of the chemical properties and mass of the target compound. Tyrocidine is an amphiphilic, cyclic peptide naturally produced by a non-ribosomal peptide synthetase (NRPS) in Bacillus brevis, with antimicrobial properties against gram-positive bacteria [1]. It functions by disrupting cell membranes, which explains its broad efficacy, but it has been shown to have hemolytic activity [2], making it unsuitable for intravenous use. Tyrocidine has been reported in the literature to have 5 main forms with the general peptide sequence of …D-Phe1/Tyr1 – Pro2 – Phe3/Trp3/Tyr3 – D-Phe4/D-Trp4/D-Tyr – Asn5 – Gln6/Asp6 – Val8 – Orn9 – Leu10… [3]

NRPS Product

Amino Acid Sequence

Molecular Formula

MW, g/mole

Tyrocidine A

F1P2F3F4N5Q6Y7V8O9L10

C66H87N13O13

1270.6619

Tyrocidine B

F1P2W3F4N5Q6Y7V8O9L10

C68H88N14O13

1309.6728

Tyrocidine C

F1P2W3W4N5Q6Y7V8O9L10

C70H89N15O13

1348.6837

Tyrocidine D

F1P2W3W4N5Q6F7V8O9L10

C72H90N16O12

1371.6997

Tyrocidine E

F1P2F3F4N5Q6F7V8O9L10

C66H87N13O12

1254.6670

Surfactin

Q1L2L3V4D5L6L7

C53H93N7O13

1036.34

Surfactin Variant

Q1L2L3V4N5L6L7

C53H94N8O12

1035.36

Surfactin has the formula C53H93N7O13 and is composed of a 7 amino acid cylic peptide with a hydrophobic fatty acid chain (Glu1 – Leu2 – D-Leu3­­ – Val4 – Asp5 – D-Leu6 – Leu7 – β-OH-C13-15) [4]. It is natively produced by Bacillus subtilis NRPS and in addition to functioning as an antibiotic it has detergent –like surfactant properties, antiviral and antifungal activities, and the capability to lyse red blood cells [5] making it another interesting candidate for a oligo-recombineering library creation.

Figure 1: cyclic peptide, Tyrocidine A 

Figure 2: Surfactin, highlighted with the blue circle at the location of the mutation from an aspartic acid residue to asparagine

Experimental Work

Tyrocidine was first detected in our native DMZ362 Brevibacillus parabrevis strain using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Initial experiments were done to develop an extraction method for the compound of interest using the DMZ362 Brevibacillus parabrevis as a positive control, Bacillus subtilis as a negative control, and an analytical standard, Tyrothricin, which contains tyrocidine A-E purified from Bacillus brevis. After testing for the compound in both the agar media and biomass, an extraction protocol was developed for use with future tyrocidine antibiotic libraries.

  1. Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.
  2. 5 mL of methanol was added to the biomass to lyse the cell membranes before they were centrifuged at 4700 x g for 5 minutes to pellet the cell material.
  3. Supernatant was carefully removed by pipetting 250 μL into an HPLC vial for analysis
  1. Biomass grown on an LB agar plate was gently scraped off the agar using a clean microscope slide (washed with ethanol or methanol before use) and placed in a labeled 15 mL centrifuge tube.
  2. 5 mL of ethyl acetate with 1% formic acid was added to the sample to ensure positively charged residues were protonated and the surfactin was extracted from the cell. Sample was sonicated for 5 minutes before it was centrifuged at 4700 x g for 5 minutes to pellet the cell material.
  3. Supernatant was carefully removed by pipetting into a fresh 15 mL Falcon tube.
  4. Ethyl acetate was evaporated with gentle N2 gas flow in a fume hood and the extract was re-dissolved in 300 μL of methanol.
  5. Methanol soluble extract was vortexed and centrifuged at 4700 x g for 5 minutes and 250 μL of sample was pipetted into an HPLC vial for analysis.
Extracted samples (in methanol) were separated by ultra-high performance liquid chromatography over an Agilent Poroshell 120 phenyl-hexyl column (2.1 x 250 mm, 2.7 μm). The detection was measured with a diode array detection coupled to quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS). Samples were run with a  a linear gradient from 10 to  100% solvent B in 15 min, followed by 100% B for 2 min and rapidly returned to 10% B to equilibrate for 3 minutes at a flow rate of 0.35 mL/min (60°C). Solvent A was Milli-Q Water, solvent B was acetonitrile and both were buffered in 20 mM formic acid. The injection volume was 1 μL. MS detection was measured on an Agilent 6545 QTOF MS with an electrospray ion source. Capillary voltage was set to 4000 V with a nozzle voltage of 500 V. Mass spectra were recorded for m/z 85–1700 in MS mode and m/z 30–1700 in MS/MS mode, with an acquisition rate of 10 spectra/s. Analysis of the spectra was done using Agilent MassHunter version B.07.00 to determine the presecnce of tyrocidine, surfactin, and modified surfactin using the “find by formula” function, with the formulas found in the table above. Manual inspection of the extracted ion chromatogram traces for [surfactin+H]+ at m/z of 1035.706 and [surfactin + Na]+ at m/z of was also performed.
Mass spectra were determined using a Bruker Autoflex II MALDI-TOF instrument. Extracted samples were spotted on an AnchorChip MALDI target plate (1 uL) and allowed to dry before being covered (1 uL) with Universal Matrix from Sigma-Aldrich (1:1 ratio of 2,5-Dihydroxybenzoic Acid to alpha-Cyano-4-hydroxycinnamic acid). The machine was operated in both linear and reflectron mode with a mass range of 500-3000. Positive and negative ion detection and reflector mode were used. Analysis of extracted samples in methanol (previously verified by HPLC-MS) by FlexAnalysis Version 3.4 indicate that tyrocidine and surfactin was clearly detectable. Surfactin was detectable in negative mode with reduced background noise compared to the positive mode, making verification a simple task.

Results

Tyrocidine HPLC-MS:

Looking at the extrapolated ion chromatogram for a mass range between 950 - 1300 Tyrocidine is clearly visible in the UHPLC-DAD-QTOFMS running under the described parameters.

Surfactin MALDI-TOF:

Surfactin HPLC-MS:

Discussion

Surfactin and Tyrosidine were detectable with MALDI-TOF after optimizing their extraction methods. Tyrocidine is not shown here, but was detectable using the positive scan mode with the reflectron. See below for detection of the exact mass/z peaks of surfactin spotted on the MALDI plates. Surfactin was detectable using the negative scan mode making for easier identification due to the lack of other compounds ionizing with that detection method.

Freshly prepared colonies that were recombineered with the previously described MAGE method to create an NRPS that could switch out the aspartic acid for asparagine, were extracted after only 12 hours of growth, and analyzed on the UHPLC-DAD-QTOFMS, showed very small amounts of detectable surfactin. This result is expected as surfactin is produced during sporulation and this occurs with older bacterial samples. In addition only about 4% of the transformants were expected to have the mutation. One sample was run that showed a mass spec correlating to the expected mass of the mutant. This result indicates that the MAGE method was successful! More samples will be submitted for verification of this result with older streaked B. subtilis mutants biomass.

References

  1. Kohli, R. M., Walsh, C. T., & Burkart, M. D. (2002). Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature, 418(6898), 658–661. doi:10.1038/nature00907
  2. Qin, C., Bu, X., Wu, X., & Guo, Z. (2003). A Chemical Approach to Generate Molecular Diversity Based on the Scaffold of Cyclic Decapeptide Antibiotic Tyrocidine A. Journal of Combinatorial Chemistry, 5(4), 353–355. doi:10.1021/cc0300255
  3. Hitzeroth, G., Vater, J., Franke, P., Gebhardt, K., & Fiedler, H.-P. (2005). Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry andin situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides. Rapid Commun. Mass Spectrom., 19(20), 2935–2942. doi:10.1002/rcm.2155
  4. Baumgart, F., Kluge, B., Ullrich, C., Vater, J., & Ziessow, D. (1991). Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy. Biochemical and Biophysical Research Communications, 177(3), 998–1005. <a href="http://dx.doi.org/10.1016/0006-291x(91)90637-m" target="_blank">doi:10.1016/0006-291x(91)90637-m</a>
  5. Singh, P., & Cameotra, S. S. (2004). Potential applications of microbial surfactants in biomedical sciences. Trends in Biotechnology, 22(3), 142–146. doi:10.1016/j.tibtech.2004.01.010
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