Difference between revisions of "Team:IISc Bangalore/Description"

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IISc Bangalore  
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             <li>Home
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ISc Bangalore
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text/javascript">
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<div class="navbar" id="top">
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<li>Home
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               <div  
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class="drop">
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                 <a  
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href="#overview">
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Overview
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                 </a>
 
                 </a>
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              </div>
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      </div>
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             </li>
 
             </li>
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            <li>Team
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               <div class="drop">
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      <li>Team
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                 <a href="">
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               <div  
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class="drop">
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                 <a  
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href="">
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                   Members
 
                   Members
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              </a>
 
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<a href="">
 
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Collaborations
 
 
                 </a>
 
                 </a>
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                <a href="">
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                  Collaborations
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            </div>
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                </a>
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             </li>
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</li>
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             <li>Project
 
             <li>Project
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              <div class="drop">
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                <a href="#description">
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      <div class="drop">
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                  Description
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                </a>
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    <a href="#description">
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          Description
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</a>
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                 <a href="">
 
                 <a href="">
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                  Design
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                </a>
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              Design
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                <a href="">
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                  Experiments
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                </a>
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</a>
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                 <a href="">
 
                 <a href="">
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              Experiments
 
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      </a>
 
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                <a
 
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href="">
 
 
                   Proof
 
                   Proof
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                </a>
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                 <a href="">
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            </a>
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                 <a  
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href="">
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                   Demo
 
                   Demo
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                </a>
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                 <a href="">
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          </a>
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                 <a  
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href="">
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                   Results
 
                   Results
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              </a>
 
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<a href="">
 
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Notebook
 
 
                 </a>
 
                 </a>
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                <a href="">
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                  Notebook
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      </div>
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                </a>
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              </div>
 
             </li>
 
             </li>
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            <li>Parts
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               <div class="drop">
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      <li>Parts
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                 <a href="">
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               <div  
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                   Basic parts
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class="drop">
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                 <a  
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href="">
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                   Basic  
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parts
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                 </a>
 
                 </a>
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                <a href="">
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                   Composite
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      <a href="">
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                </a>
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                <a href="">
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                  Part collection
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Composite
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                 </a>
 
                 </a>
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          <a href="">
 
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  Part collection
 
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               </div>
 
               </div>
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</li>
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             <li>Safety</li>
 
             <li>Safety</li>
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            <li>Attributions</li>
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            <li>Practices</li>
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          <li>Attributions</li>
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            <li>Awards</li>
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      <li>Practices</li>
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<li>Awards</li>
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           </ul>
 
           </ul>
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        </div>
  
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        <div class="content">
  
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        <div
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              <span class="cellfiefuge">
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                Cellfiefuge
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              </span>
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              <p class="tagline">
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                An auto-pelleting system
 +
              </p>
  
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class="content">
 
  
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<span class="cellfiefuge">
 
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      Cellfiefuge
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               <svg height="60" width="320" xmlns="http://www.w3.org/2000/svg">
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                  <a href="#description">Description</a>
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class="tagline">
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                An
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auto-pelleting system
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height="60" width="320"  
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        <div class="boxes" id="description">
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          <p class="header">Description</p>
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          <div class="bara_dabba">
  
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            <div class="semi_dabba" id="first">
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id="description">
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                <p class="glimpse">
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          <p
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                 <a href="#overview">Overview</a><br><br>
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class="header">Description</p>
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                 </p>
 
                 </p>
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             </div>
 
             </div>
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            <div class="semi_dabba" id="second">
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              <div class="chotta_dabba">
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                <p class="glimpse">
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                  <a href="#design">Design</a><br><br>
  
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    <div class="semi_dabba"
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                 </p>
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               </div>
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id="second">
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              </p>
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             </div>
 
             </div>
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           </div>
  
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          <div class="bara_dabba">
  
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          <div  
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            <div class="semi_dabba" id="third">
 +
              <div class="chotta_dabba">
 +
                <p class="glimpse">
 +
                  <a href="#protein">Protein production module</a><br><br>
  
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class="bara_dabba">
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                 </p>
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<div class="semi_dabba" id="third">
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class="chotta_dabba">
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<p class="glimpse">
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  <a href="#protein">Protein
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production module</a><br><br>
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          </p>
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               </div>
 
               </div>
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            </div>
 +
            <div class="semi_dabba" id="fourth">
  
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          </div>
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              <div class="chotta_dabba">
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            <div
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                <p class="glimpse">
 +
                  <a href="#aggre">Auto Aggregation module</a><br><br>
  
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class="semi_dabba" id="fourth">
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                 </p>
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  <a href="#aggre">Auto Aggregation
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module</a><br><br>
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</p>
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               </div>
 
               </div>
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            </div>
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</div>
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           </div>
 
           </div>
  
  
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         </div>
 +
        <div id="sidebar" class="sidebar">
  
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</div>
+
          <a href="javascript:void(0)" class="closebtn" onclick="closeBar()">&times;</a>
−
        <div id="sidebar"  
+
          <a href="#overview">Overview</a>
 +
          <a href="#design">Design</a>
 +
          <a href="#protein">Protein</a>
 +
          <a href="#aggre">Aggregation</a>
 +
          <a href="#ref">References</a>
 +
          <a href="#top">Back to top</a>
  
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class="sidebar">
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               <a href="https://facebook.com/iiscbangaloreigem/" >
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             Overview
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          <div class="text">
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            <p>Since the advent of rDNA technology in the late 1970s, the use of recombinant protein has been on the rise, starting from insulin and hormones all the way through to monoclonal antibodies and one hundred and fifty-one FDA approved protein based recombinant pharmaceuticals by the year 2009[1]. Despite being in high demand, recombinant proteins have heavy price tags, thanks to the significant manufacturing costs. One of the important steps in the manufacturing process is Centrifugation but on an industrial scale, it is an economic nightmare. [2] Further, existing industrial setups use complex sensors to monitor cell density to determine the point at which to begin separation of the cells from the cell culture. One can immediately see the humongous relief that would be associated with finding a way to deal with the above problems. Hence, we came up with the idea of our project, the Cellfifuge.</p>
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Overview
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<div class="text">
 
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<p>Since the advent of rDNA
 
−
 
−
technology in the late 1970s, the
 
−
 
−
use of recombinant protein has been
 
−
 
−
on the rise, starting from insulin
 
−
 
−
and hormones all the way through to
 
−
 
−
monoclonal antibodies and one
 
−
 
−
hundred and fifty-one FDA approved
 
−
 
−
protein based recombinant
 
−
 
−
pharmaceuticals by the year 2009[1].
 
−
 
−
Despite being in high demand,
 
−
 
−
recombinant proteins have heavy
 
−
 
−
price tags, thanks to the
 
−
 
−
significant manufacturing costs. One
 
−
 
−
of the important steps in the
 
−
 
−
manufacturing process is
 
−
 
−
Centrifugation but on an industrial
 
−
 
−
scale, it is an economic nightmare.
 
−
 
−
[2] Further, existing industrial
 
−
 
−
setups use complex sensors to
 
−
 
−
monitor cell density to determine
 
−
 
−
the point at which to begin
 
−
 
−
separation of the cells from the
 
−
 
−
cell culture. One can immediately
 
−
 
−
see the humongous relief that would
 
−
 
−
be associated with finding a way to
 
−
 
−
deal with the above problems. Hence,
 
−
 
−
we came up with the idea of our
 
−
 
−
project, the Cellfifuge.</p>
 
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  </div>
 
 
         </div>
 
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<svg class="panel" onclick="openBar()" enable-background="new 0 0 128 128" height="40px" id="Layer_1" version="1.1" viewBox="0 0 128 128" width="40px" xml:space="preserve" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g><g><path d="M64,0C28.656,0,0,28.656,0,64s28.656,64,64,64s64-28.656,64-64S99.344,0,64,0z M64,120    C33.125,120,8,94.875,8,64S33.125,8,64,8s56,25.125,56,56S94.875,120,64,120z" fill="#B0BEC5"/></g></g><g><g><path d="M96,56H72V32c0-4.414-3.586-8-8-8s-8,3.586-8,8v24H32c-4.414,0-8,3.586-8,8s3.586,8,8,8h24v24    c0,4.414,3.586,8,8,8s8-3.586,8-8V72h24c4.414,0,8-3.586,8-8S100.414,56,96,56z" fill="#8BC34A"/></g></g></svg>
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           Our Bioreactor Design
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           Our  
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Bioreactor Design
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           </div>
 
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          <div id="left" class="text">
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            <p>We realized that manufacturing units use expensive and energy expensive machinery [3]. Hence, we engineered our bacteria in such a way that it could do most of the work done by these monstrous machines so that we no longer have to buy them and pay their bills.
−
       
+
To increase the yield of the product, we came up with the idea of separating cell growth and target protein production so that when the cells have grown up to a high enough cell density, they shuttle all their energy towards the required protein production.[4] Based on the above ideas, we came up with the design of our bioreactor. (Figure 1)</p>
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<div id="left" class="text">
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    <p>We realized that  
+
−
 
+
−
manufacturing units use expensive  
+
−
 
+
−
and energy expensive machinery [3].  
+
−
 
+
−
Hence, we engineered our bacteria in  
+
−
 
+
−
such a way that it could do most of  
+
−
 
+
−
the work done by these monstrous  
+
−
 
+
−
machines so that we no longer have  
+
−
 
+
−
to buy them and pay their bills.
+
−
To  
+
−
 
+
−
increase the yield of the product,  
+
−
 
+
−
we came up with the idea of  
+
−
 
+
−
separating cell growth and target  
+
−
 
+
−
protein production so that when the  
+
−
 
+
−
cells have grown up to a high enough  
+
−
 
+
−
cell density, they shuttle all their  
+
−
 
+
−
energy towards the required protein  
+
−
 
+
−
production.[4] Based on the above  
+
−
 
+
−
ideas, we came up with the design of  
+
−
 
+
−
our bioreactor. (Figure 1)</p>
+
−
 
+
−
<p>We realized that we would not be
+
−
 
+
−
able to work on the whole idea this
+
−
 
+
−
summer so, we focussed on two
+
−
 
+
−
modules of our design: the Protein
+
−
 
+
−
production module and the
+
−
 
+
−
auto-aggregation module leaving the
+
−
 
+
−
arresting of cell growth for future
+
−
 
+
−
iGEM teams as a seed for their
+
  
−
ideas.</p>
+
<p>We realized that we would not be able to work on the whole idea this summer so, we focussed on two modules of our design: the Protein production module and the auto-aggregation module leaving the arresting of cell growth for future iGEM teams as a seed for their ideas.</p>
  
 
           </div>
 
           </div>
−
       
+
          <div >
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+
             <img class="image" src="designa.jpg" />Figure 1: The bioreactor design of Cellfifuge
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  <div >
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             <img  
+
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+
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class="image" src="designa.jpg"  
+
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+
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/>Figure 1: The bioreactor design of  
+
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+
−
Cellfifuge
+
 
           </div>
 
           </div>
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<svg class="panel" onclick="openBar()" enable-background="new 0 0 128 128" height="40px" id="Layer_1" version="1.1" viewBox="0 0 128 128" width="40px" xml:space="preserve" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g><g><path d="M64,0C28.656,0,0,28.656,0,64s28.656,64,64,64s64-28.656,64-64S99.344,0,64,0z M64,120    C33.125,120,8,94.875,8,64S33.125,8,64,8s56,25.125,56,56S94.875,120,64,120z" fill="#B0BEC5"/></g></g><g><g><path d="M96,56H72V32c0-4.414-3.586-8-8-8s-8,3.586-8,8v24H32c-4.414,0-8,3.586-8,8s3.586,8,8,8h24v24    c0,4.414,3.586,8,8,8s8-3.586,8-8V72h24c4.414,0,8-3.586,8-8S100.414,56,96,56z" fill="#8BC34A"/></g></g></svg>
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        </span>
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<svg class="panel"  
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         <div id="protein" class="sub">
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           Protein Production Module
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         <div id="protein"  
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           <div  
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class="header">
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           Protein  
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Production Module
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           </div>
 
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          <div class="text">
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+
            <p>Induction synthesis of protein of interest is another important step. Microorganisms in bioreactors are induced at the OD, at which the protein production is maximum. [5] Because of this, factories install sophisticated machinery to keep track of the OD value of the culture so as to know when to induce the production. We wanted our bacteria to know when to start protein production and start it by itself. Hence, we used the lux quorum sensing system to sense this cell density. [6] We have expressed the gene for our protein of interest under the luxI gene so that protein of interest is produced in significant quantities only when the quorum is achieved.(Figure 2) We also wish to tune the quorum sensing system by tweaking with the strength of the promoters involved in quorum sensing.
−
        <div class="text">
+
−
         
+
−
 
+
−
  <p>Induction synthesis of protein  
+
−
 
+
−
of interest is another important  
+
−
 
+
−
step. Microorganisms in bioreactors  
+
−
 
+
−
are induced at the OD, at which the  
+
−
 
+
−
protein production is maximum. [5]  
+
−
 
+
−
Because of this, factories install  
+
−
 
+
−
sophisticated machinery to keep  
+
−
 
+
−
track of the OD value of the culture  
+
−
 
+
−
so as to know when to induce the  
+
−
 
+
−
production. We wanted our bacteria  
+
−
 
+
−
to know when to start protein  
+
−
 
+
−
production and start it by itself.  
+
−
 
+
−
Hence, we used the lux quorum  
+
−
 
+
−
sensing system to sense this cell  
+
−
 
+
−
density. [6] We have expressed the  
+
−
 
+
−
gene for our protein of interest  
+
−
 
+
−
under the luxI gene so that protein  
+
−
 
+
−
of interest is produced in  
+
−
 
+
−
significant quantities only when the  
+
−
 
+
−
quorum is achieved.(Figure 2) We  
+
−
 
+
−
also wish to tune the quorum sensing  
+
−
 
+
−
system by tweaking with the strength  
+
−
 
+
−
of the promoters involved in quorum  
+
−
 
+
−
sensing.
+
 
             </p>
 
             </p>
−
         
+
            <img class="image" src="lux.jpg" />Figure 2 : The lux quorum sensing system. Note the gene of interest "geneX*" downstream of the luxI gene. Image Courtesy: ETH Zurich 2013, iGEM team.
−
 
+
          </div>
−
<img class="image" src="lux.jpg"  
+
−
 
+
−
/>Figure 2 : The lux quorum sensing  
+
−
 
+
−
system. Note the gene of interest  
+
−
 
+
−
"geneX*" downstream of the luxI  
+
−
 
+
−
gene. Image Courtesy: ETH Zurich  
+
−
 
+
−
2013, iGEM team.
+
 
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         <div id="aggre" class="sub">
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           Auto-aggregation Module
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To achieve cellular aggregation, we use the famous auto-transporter protein, Antigen43 (Ag43). [7] It has shown to cause auto-aggregation of cells when overexpressed. (Figure 3) We plan to exploit this property of Ag43 to substitute centrifugation. We acknowledge previous iGEM teams who have worked on Ag43 (Hokkaido University, 2012; Aberdeen Scotland, 2014 and some more) as we got the BioBricks for Ag43 readily available.  We intend to express Ag43 only when the cells have made the product of interest. Hence, we use the diauxic shift in the media to act as a switch for the expression of Ag43.
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To achieve cellular  
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             <img class="image" src="tube.jpg" />Figure 3: a) Cell culture without expression of Ag43 b) Cell culture overexpressing Ag43
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          <li> Medipally S.R., Yusoff F. Md., Banerjee S., Shariff M. Microalgae as sustainable renewable energy Feedstock for Biofuel Production, BioMed Research International, 2015. </li>
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          <li> Motoo Suzuki, Rohini Roy, Haiyan Zheng, Nancy Woychik, Masayori Inouye, Bacterial Bioreactors for High Yield Production of Recombinant Protein. The Journal of Biological Chemistry, 2006.</li>
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          <li> Miller M.B., Bassler B.L., Quorum Sensing in Bacteria, Annual Review of Microbiology, 2001.</li>
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Revision as of 16:15, 30 June 2016

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Cellfiefuge

An auto-pelleting system

Description
Overview

Since the advent of rDNA technology in the late 1970s, the use of recombinant protein has been on the rise, starting from insulin and hormones all the way through to monoclonal antibodies and one hundred and fifty-one FDA approved protein based recombinant pharmaceuticals by the year 2009[1]. Despite being in high demand, recombinant proteins have heavy price tags, thanks to the significant manufacturing costs. One of the important steps in the manufacturing process is Centrifugation but on an industrial scale, it is an economic nightmare. [2] Further, existing industrial setups use complex sensors to monitor cell density to determine the point at which to begin separation of the cells from the cell culture. One can immediately see the humongous relief that would be associated with finding a way to deal with the above problems. Hence, we came up with the idea of our project, the Cellfifuge.

Our Bioreactor Design

We realized that manufacturing units use expensive and energy expensive machinery [3]. Hence, we engineered our bacteria in such a way that it could do most of the work done by these monstrous machines so that we no longer have to buy them and pay their bills. To increase the yield of the product, we came up with the idea of separating cell growth and target protein production so that when the cells have grown up to a high enough cell density, they shuttle all their energy towards the required protein production.[4] Based on the above ideas, we came up with the design of our bioreactor. (Figure 1)

We realized that we would not be able to work on the whole idea this summer so, we focussed on two modules of our design: the Protein production module and the auto-aggregation module leaving the arresting of cell growth for future iGEM teams as a seed for their ideas.

Figure 1: The bioreactor design of Cellfifuge
Protein Production Module

Induction synthesis of protein of interest is another important step. Microorganisms in bioreactors are induced at the OD, at which the protein production is maximum. [5] Because of this, factories install sophisticated machinery to keep track of the OD value of the culture so as to know when to induce the production. We wanted our bacteria to know when to start protein production and start it by itself. Hence, we used the lux quorum sensing system to sense this cell density. [6] We have expressed the gene for our protein of interest under the luxI gene so that protein of interest is produced in significant quantities only when the quorum is achieved.(Figure 2) We also wish to tune the quorum sensing system by tweaking with the strength of the promoters involved in quorum sensing.

Figure 2 : The lux quorum sensing system. Note the gene of interest "geneX*" downstream of the luxI gene. Image Courtesy: ETH Zurich 2013, iGEM team.
Auto-aggregation Module

To achieve cellular aggregation, we use the famous auto-transporter protein, Antigen43 (Ag43). [7] It has shown to cause auto-aggregation of cells when overexpressed. (Figure 3) We plan to exploit this property of Ag43 to substitute centrifugation. We acknowledge previous iGEM teams who have worked on Ag43 (Hokkaido University, 2012; Aberdeen Scotland, 2014 and some more) as we got the BioBricks for Ag43 readily available. We intend to express Ag43 only when the cells have made the product of interest. Hence, we use the diauxic shift in the media to act as a switch for the expression of Ag43.

Figure 3: a) Cell culture without expression of Ag43 b) Cell culture overexpressing Ag43 Source: MarjanW. van derWoude et al, 2008
References
  1. Ferrer-Miralles N, Domingo-Espin J, Corchero JL, Vazquez E, Villaverde A. Microbial factories for recombinant pharmaceuticals, Microbial Cell Factories, 2009:8:17.
  2. Medipally S.R., Yusoff F. Md., Banerjee S., Shariff M. Microalgae as sustainable renewable energy Feedstock for Biofuel Production, BioMed Research International, 2015.
  3. Felo M., Christensen B., Higgins J., Process cost and facility considerations in the selection of primary cell culture clarification technology, AIChE, 2013.
  4. Motoo Suzuki, Rohini Roy, Haiyan Zheng, Nancy Woychik, Masayori Inouye, Bacterial Bioreactors for High Yield Production of Recombinant Protein. The Journal of Biological Chemistry, 2006.
  5. Rosano G.L., Ceccarelli E. A., Recombinant protein expression in Escherichia coli: advances and challenges, Frontiers in Microbiology, 2014.
  6. Miller M.B., Bassler B.L., Quorum Sensing in Bacteria, Annual Review of Microbiology, 2001.
  7. Marjan W. van der Woude, Ian R. Henderson, Regulation and Function of Ag43 (Flu), Annu. Rev. Microbiol., 2008.