Difference between revisions of "Team:NortheasternU-Boston/Design"

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{{NortheasternU-Boston}}
 
{{NortheasternU-Boston}}
 
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      <div class="row jumbo">
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        </html>{{NortheasternU-Boston-Sidebar}}<html>
  
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        <div class="col-sm-9 home-abstract no-float">
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<div class="column full_size">
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          <div class="header">
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<h1>Design</h1>
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            <h1>Project Design</h1>
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          </div>
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          <div class="content-text">
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<h3>Expression without Cloning!</h3>
 
<p>
 
<p>
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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A key advantage of cell-free systems is that they allow expression directly
 +
from linear amplified DNA rather than from a bacterial plasmid. This enabled
 +
our overall part design scheme of  IDT synthesized G-blocks containing each
 +
of our AMPs between our desired 5’UTR and Terminator, allowing us to express
 +
these AMPs after a single round of PCR with overhanging primers containing
 +
promoter regions. This schema allowed us to test numerous AMPs as well as GFP
 +
and methionine aminopeptidase under the control of two promoters without
 +
having to go through the time consuming process of cloning a bacterial plasmid
 +
for each expression construct.
 
</p>
 
</p>
  
 
<p>
 
<p>
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:NortheasternU-Boston/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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This allows for an extremely iterative design process where a part can be conceived of,
 +
a G-block synthesized, a PCR amplification performed, and a part tested in a
 +
cell-free expression system within a matter of two weeks.
 
</p>
 
</p>
  
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</div>
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<img src="https://static.igem.org/mediawiki/2017/c/c2/T--NortheasternU-Boston--DesignBlocks.png"
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width="95%" style="max-width:100%;margin:10px"></img>
  
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<div class="column half_size">
 
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<h5>What should this page contain?</h5>
 
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<ul>
 
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<li>Explanation of the engineering principles your team used in your design</li>
 
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<li>Discussion of the design iterations your team went through</li>
 
−
<li>Experimental plan to test your designs</li>
 
−
</ul>
 
  
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</div>
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<h3>Experimental Plans</h3>
 +
<p>
 +
We set out to answer key questions concerning what level of complexity of AMP
 +
could be produced in our cell-free system. We also wanted to discover whether
 +
or not we could rescue AMP function through treatment with methionine aminopeptidase
 +
in the case where a non-canonical N-terminal methionine was added to the peptide
 +
as a start codon for expression. AMP effectivity is gauged via bacterial killing
 +
effectivity measured by OD600 growth curves or various other assays.
 +
Our experimental design would then be to test our panel of AMPs across promoters
 +
and in the presence or absence of methionine aminopeptidase also produced via
 +
cell-free reactions. Pardee 2016 reported that the AMPs that they produced in
 +
cell-free reactions were equally viable in purified and unpurified form, therefore
 +
purification was a step that we avoided in order to create a streamlined workflow
 +
directly from synthesis to amplification to expression to characterization.
 +
</p>
  
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<div class="column half_size">
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<h3>Citations</h3>
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<h5>Inspiration</h5>
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<ul>
 
<ul>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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  <li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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    <p>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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Pardee, K., Slomovic, S., Nguyen, P. Q., Lee, J. W., Donghia, N., Burrill, D., . . . Collins, J. J. (2016). Portable, On-Demand Biomolecular Manufacturing. Cell, 167(1), 248-259.e212. doi:<a href="https://doi.org/10.1016/j.cell.2016.09.013" target="_blank">https://doi.org/10.1016/j.cell.2016.09.013</a>
 +
<a href="http://www.sciencedirect.com.ezproxy.neu.edu/science/article/pii/S0092867416312466" target="_blank">http://www.sciencedirect.com.ezproxy.neu.edu/science/article/pii/S0092867416312466</a>
 +
    </p>
 +
  </li>
 
</ul>
 
</ul>
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</div>
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          </div>
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        </div>
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      </div>
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      <div class="row neu-igem-footer">
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  <!-- SPONSOR LOGOS -->
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  <div class="col-xs-12">
  
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    <!-- don't call these classes "sponsor" since adblock will destroy it -->
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    <div class="neusponsor-container">
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      <div class="neusponsor-header">
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        <span class="neusponsor-title">Our Generous Sponsors</span>
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      </div>
 +
      <hr/>
 +
      <ul class="neusponsor-list">
 +
        <li class="neusponsor-item">
 +
          <a href="http://compasstherapeutics.com" class="neusponsor-item-link" target="_blank"
 +
            title="Compass Therapeutics">
 +
            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/3/33/T--NortheasternU-Boston--SponsorCompass.png"
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              alt="Compass Therapeutics"/>
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          </a>
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        </li>
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        <li class="neusponsor-item">
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          <a href="https://www.idtdna.com/site" class="neusponsor-item-link" target="_blank"
 +
            title="IDT Integrated DNA Technologies">
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            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/8/84/T--NortheasternU-Boston--SponsorIDT.png"
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              alt="IDT Integrated DNA Technologies"/>
 +
          </a>
 +
        </li>
 +
        <li class="neusponsor-item">
 +
          <a href="https://www.neb.com" class="neusponsor-item-link" target="_blank"
 +
            title="NEB New England Biolabs">
 +
            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/d/d7/T--NortheasternU-Boston--SponsorNEB.png"
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              alt="NEB New England Biolabs"/>
 +
          </a>
 +
        </li>
 +
        <li class="neusponsor-item">
 +
          <a href="http://www.northeastern.edu/cos/" class="neusponsor-item-link" target="_blank"
 +
            title="Northeastern University College of Science">
 +
            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/5/5e/T--NortheasternU-Boston--SponsorNEUCoS.png"
 +
              alt="Northeastern University College of Science"/>
 +
          </a>
 +
        </li>
 +
        <li class="neusponsor-item">
 +
          <a href="http://www.bioe.neu.edu" class="neusponsor-item-link" target="_blank"
 +
            title="Northeastern Univeristy Department of Bioengineering">
 +
            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/7/7d/T--NortheasternU-Boston--SponsorNEUBioE.png"
 +
              alt="Northeastern University Department of Bioengineering"/>
 +
          </a>
 +
        </li>
 +
        <li class="neusponsor-item">
 +
          <a href="http://www.che.neu.edu" class="neusponsor-item-link" target="_blank"
 +
            title="Northeastern University Department of Chemical Engineering">
 +
            <img class="neusponsor-img" src="https://static.igem.org/mediawiki/2017/3/3a/T--NortheasternU-Boston--SponsorNEUChemE.png"
 +
              alt="Northeastern University Department of Chemical Engineering"/>
 +
          </a>
 +
        </li>
  
 +
      </ul>
 +
    </div>
 +
 +
  </div>
 +
</div>
  
 +
    </div>
 +
  </div>
 
</html>
 
</html>

Revision as of 19:18, 1 November 2017

Project Design

Expression without Cloning!

A key advantage of cell-free systems is that they allow expression directly from linear amplified DNA rather than from a bacterial plasmid. This enabled our overall part design scheme of IDT synthesized G-blocks containing each of our AMPs between our desired 5’UTR and Terminator, allowing us to express these AMPs after a single round of PCR with overhanging primers containing promoter regions. This schema allowed us to test numerous AMPs as well as GFP and methionine aminopeptidase under the control of two promoters without having to go through the time consuming process of cloning a bacterial plasmid for each expression construct.

This allows for an extremely iterative design process where a part can be conceived of, a G-block synthesized, a PCR amplification performed, and a part tested in a cell-free expression system within a matter of two weeks.

Experimental Plans

We set out to answer key questions concerning what level of complexity of AMP could be produced in our cell-free system. We also wanted to discover whether or not we could rescue AMP function through treatment with methionine aminopeptidase in the case where a non-canonical N-terminal methionine was added to the peptide as a start codon for expression. AMP effectivity is gauged via bacterial killing effectivity measured by OD600 growth curves or various other assays. Our experimental design would then be to test our panel of AMPs across promoters and in the presence or absence of methionine aminopeptidase also produced via cell-free reactions. Pardee 2016 reported that the AMPs that they produced in cell-free reactions were equally viable in purified and unpurified form, therefore purification was a step that we avoided in order to create a streamlined workflow directly from synthesis to amplification to expression to characterization.

Citations

Our Generous Sponsors