Difference between revisions of "Team:UCopenhagen/Protocols"

Line 6: Line 6:
  
 
<!-- Header -->
 
<!-- Header -->
−
     <a name="project-page"></a>
+
     <a name="project"></a>
 
     <div class="intro-header4">
 
     <div class="intro-header4">
 
         <div class="container">
 
         <div class="container">
Line 14: Line 14:
 
                     <div class="intro-message2">
 
                     <div class="intro-message2">
 
<h3></h3>
 
<h3></h3>
 +
                        <h1>P R O T O C O L S</h1> 
 
<h3></h3>
 
<h3></h3>
−
<h3></h3>
 
−
<h3></h3>
 
−
<h3></h3>
 
−
                        <h1>P R O T O C O L S</h1>
 
−
                        <h3></h3>
 
−
           
 
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
Line 27: Line 22:
 
         </div>
 
         </div>
 
         <!-- /.container -->
 
         <!-- /.container -->
−
 
−
    </div>
 
−
    <!-- /.intro-header -->
 
−
 
−
 
 
<div class="content-section-a">
 
<div class="content-section-a">
 
<div class="container">
 
<div class="container">
−
             <div class="row">
+
             <div>
−
                <div class="col-lg-5 col-sm-6">
+
 
                     <hr class="section-heading-spacer">
 
                     <hr class="section-heading-spacer">
 
                     <div class="clearfix"></div>
 
                     <div class="clearfix"></div>
−
                     <h2 class="section-heading">Introduction </h2>
+
                     <h2 class="section-heading">Experiments</h2>
−
                     <p class="lead">Our team believes that establishing a stable platform for scientists to create naïve orthogonal living compartments, would allow for an unpredictable advancement in the field of synthetic biology. Our project will not attempt to create an endosymbiont, but instead investigate the mechanisms in free-living cells in a bottom-up approach to endosymbiosis.
+
                     <p class="lead">
−
The endosymbiotic theory, formulated in the early years of the previous century, outlines that the organelles of the eukaryotic cell, such as the mitochondria, have their origin in free-living prokaryotes engulfed by bigger cells. These incorporated cells then co-evolved with their host conferring to it novel emergent properties which ultimately helped fuel the development of more complex multicellular biological systems such as plants and animals (Archibald, 2015). </p>
+
<strong>Amplification of genes:</strong>
−
 
+
<ul style="text-align:left; color:white;">
 +
<li>Synthetically produce a codon-optimised version of yddG. </li>
 +
<li>Amplify trpE and aroG from WT E.coli MG1655</li>
 +
<li> Point mutations for feedback resistance in trpE and aroG: Using primers with overhangs containing point mutations, and splitting the gene in two, then combining the two parts when inserting in the expression vector. </li>
 +
<li> Created vector with combinations of one, two and three genes in the USER casette, using primers with overhangs.</li>
 +
</ul>
 +
</p>
 +
<p class="lead">
 +
To make the point mutations for trpE and aroG, two sets of vectors for each gene was designed (illustration). Overhangs in the end of the primers enable USER cassette insertion, while the primer overhangs in the center contain a point mutation. When the two parts are being amplified individually, the transformation into vector in expression host will be done with USER ligation.
 +
<br><br>
 +
<strong>Vector design</strong>
 +
<br><br>
 +
Protein import. USER casette and His tag. <br>
 +
Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.
 +
<br><br>
 +
<strong>Expression and production</strong>
 +
<ul style="text-align:left; color:white;">
 +
<li>Expression checked with Western blotting: all genes HIS tagged. </li>
 +
<li>Tryptophane production by E.coli with one, two or three genes, and nuder different levels of inducing agents evaluated on HPLC</li>
 +
</ul>
 +
</p>
 +
<p class="lead">
 +
<strong>Co-growth of E.coli and yeast</strong>
 +
<ul style="text-align:left; color:white;">
 +
<li>Find yeast minimal media where E.coli can grow</li>
 +
<li>Grow yeast in same media after E.coli has grown, in order to establish possibility for relationship.</li>
 +
<li>Grow tryptophane producing E.coli for different time periods, then remove them and grow auxotrophic yeast in the media containing the produced tryptophane. </li>
 +
</ul>
 +
</p>
 +
<p class="lead">
 +
<strong>Growth of E.coli and yeast in same minimal yeast medium</strong>
 +
<br><br>
 +
E. coli strains MG1655 and BL21 were grown in several media in order to find a minimal yeast media where E.coli could survive. With inspiration from (van Summeren-Wesenhagen and Marienhagen, 2014), we decided to grow E.coli in the minimal yeast media YNB with the pH adjusted to 7 instead of 4 as original.
 
<br>
 
<br>
−
 
+
After ON growth of E.coli in YNB pH 7, the media was cleared of E.coli by spinning and filtration, after which it was inoculated with yeast (AM94), to ensure that the E.coli does not produce substances hindering yeast growth (protocol).<br>
−
<p>We have identified three mechanisms we believe to be mandatory for the development of a stable endosymbiotic relationship, which we will be trying to replicate in free-living cells. First of all, in order for the relationship to be stable, the two organisms must  be mutually dependent on each other; there must be a mutually beneficial interaction between host and symbiont. Secondly, there has to be some sort of control and synchronization of symbiont replication. If the symbiont were to be replicating freely we could end up with way too many or not enough symbionts in the host.  Finally, a common feature of the endosymbiotic organelles we have looked at, is the transfer of genes from the symbiont to the host. Because of this transfer, the gene and protein expression is taking place in the nucleus and the proteins and metabolites are transported to the organelle. This import of proteins is interesting not just for understanding endosymbiosis, but also for the potential applications in synthetic biology.</p>
+
This experiment is a prerequisite for our next experiment.
−
 
+
<br><br>
−
<br>
+
<strong>Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli</strong>
−
 
+
<br><br>
−
<p>Based on these considerations, we decided to work on three distinct, but intertwined, projects pertaining to endosymbiosis, namely Interdependence, Number Control, and Protein import. We believe that by combining these three projects, a key step towards the understanding of endosymbiosis and its employment in synthetic biology will be obtained. </p>
+
This experiment utilizes the same protocol as the previous (protocol), but now in YNB pH7 media without a tryptophan source, with tryptophan overproducing E.coli and with a tryptophan auxotrophic yeast strain.This experiment is performed with single, double and triple transformations: That is, E.coli with trpE(fbr), aroG(fbr) and yddG alone or in combinations. The growth of yeast is measured using OD600 measurements to evaluate the successful complementation of the yeast amino acid auxotrophy by E.coli tryptophan production.
−
                </div>
+
              </p>
−
                <div class="col-lg-5 col-lg-offset-2 col-sm-6">
+
−
                    <img class="img-responsive" src="img/national-logo.jpg" alt="">
+
−
                </div>
+
−
            </div>
+
−
 
+
−
        </div>
+
−
        <!-- /.container -->
+
−
 
+
 
     </div>
 
     </div>
 +
</div>
 
</div>
 
</div>
 
          
 
          
−
    <div class="content-section-b">
+
<div class="content-section-b">
−
        <div class="container">
+
<div class="container">
−
             <div class="row">
+
             <div>
−
                <div class="col-lg-5 col-lg-offset-1 col-sm-push-6  col-sm-6">
+
 
                     <hr class="section-heading-spacer">
 
                     <hr class="section-heading-spacer">
 
                     <div class="clearfix"></div>
 
                     <div class="clearfix"></div>
−
                     <h2 class="section-heading">Applications and Implications</h2>
+
                     <h2 class="section-heading">Design process</h2>
−
                    <<p>By understanding the basic principles behind the creation of stable endosymbiotic events we hope that in the future it will be possible to use artificial endosymbiosis as a new technology in synthetic biology, and we believe that value can be created in the foundational track of the iGEM competition. History has shown that great scientific advances has followed the implementation of new revolutionary technologies (Gershon 2003). </p>
+
<p class="lead">
−
<br>
+
<br><br>
−
<p>We envision that artificial endosymbiosis could be applied in a broad range of fields, including agriculture, medicine and production of valuable compounds. A deeper understanding of the relationships intertwining endosymbionts and their hosts could unravel new knowledge applicable for the treatment of mitochondrial diseases, while a living compartment able to fixate nitrogen from the air could decrease the fertilizer use in agricultural production. </p>
+
In our design process, we have considered a wide range of possible gene combinations. Genes that when over-expressed would have the greatest impact were chosen. This is due to the time constraints set and simplicity. Initially, we considered simply overexpressing the tryptophane operon, but quickly realised this would be highly downregulated due to negative feedback regulation.  
−
<br>
+
<br><br>
−
<p>However, the applications are only limited by the imagination of future users. Indeed, the game-changing role of endosymbiosis has not gone unseen to the eyes of the modern bioengineers, who predict that the establishment of a novel interaction has the potential to radically alter the host cell physiology without directly affecting the host genome (Scientific America Vol 105 pp. 36-45).</p>
+
We decided that an exporter would be beneficial by reducing the intracellular Trp concentration, which would release the feedback regulation. We also thought of deleting endogenous trpR, but making such a deletion would make our project overly complicated due to the difficulty of making such a deletion is E.coli.</p>
−
<br>
+
                  </div>
−
<p>Before the potential application of artificial endosymbiosis, there are many things to consider. While the current regulations regarding GMO limits what is possible to apply in agriculture and medicine, regulations regarding synthetically modified organisms (SMOs) have not yet been systematically put into place. How will a new field of SMO be regulated, and how will it influence possible applications of artificial endosymbiosis?</p>
+
−
<br>
+
−
<p>In addition to our scientific investigation we are enthused to trigger debate about synthetic biology. We intend to podcast intriguing conversations with experts, thereby hoping to reach the general public and impel the discussion about the ethics and future prospects in combining biology and engineering.</p>
+
−
                     
+
−
                    </div>
+
−
                    <div class="col-lg-6 col-sm-pull-6  col-sm-6">
+
−
                   
+
−
                        <img class="img-responsive2" src="img/Lacrosse2.jpg" alt="">
+
−
                    </div>
+
 
                 </div>
 
                 </div>
 
                 </div>
 
                 </div>
−
            </div>
 
−
</div>
 
−
 
 
−
<a  name="socialmeida"></a>
 
−
    <div class="banner">
 
  
 +
<a  name="socialmedia"></a>
 
         <div class="container">
 
         <div class="container">
−
 
+
          <div class="row">
−
            <div class="row">
+
                 <div class="col-lg-5">
−
                 <div class="col-lg-6">
+
                     <h2>Find Incell here:</h2>
−
                     <h2>Find inCell here:</h2>
+
 
                 </div>
 
                 </div>
−
                 <div class="col-lg-6">
+
                 <div class="col-lg-7">
 +
<br>
 
                     <ul class="list-inline banner-social-buttons">
 
                     <ul class="list-inline banner-social-buttons">
 
                         <li>
 
                         <li>
Line 114: Line 114:
 
         </div>
 
         </div>
 
         <!-- /.container -->
 
         <!-- /.container -->
 +
    </div>
 +
    <!-- /.intro-header -->
  
 
     </div>
 
     </div>
−
                   
 
 
     <!-- /.banner -->
 
     <!-- /.banner -->
 
     <!-- Interactive bar -->
 
     <!-- Interactive bar -->
Line 127: Line 128:
 
                     <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
 
                     <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
 
                     <li>
 
                     <li>
−
                 <a class="page-scroll" href="#">Top</a>
+
                 <a class="page-scroll" href="#Top">Introduction</a>
−
</li>
+
                    </li>
 +
                    <li>
 +
                <a class="page-scroll" href="#Design">Final design</a>
 +
                    </li>
 +
                    <li>
 +
                <a class="page-scroll" href="#experiment">Experiments</a>
 +
                    </li>
 +
                    <li>
 +
                <a class="page-scroll" href="#process">Design Process</a>
 +
                    </li>
 +
 
 
                 </ul>
 
                 </ul>
 
             </div>
 
             </div>
Line 137: Line 148:
 
                     <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
 
                     <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
 
                     <li>
 
                     <li>
−
                         <a class="page-scroll" href="https://2017.igem.org/Team:UCopenhagen/Notebook">Previous</a>
+
                         <a class="page-scroll" href="https://2017.igem.org/Team:UCopenhagen/Project">Previous</a>
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         <a class="page-scroll" href="https://2017.igem.org/Team:UCopenhagen/Team">Next</a>
+
                         <a class="page-scroll" href="https://2017.igem.org/Team:UCopenhagen/Number-Control">Next</a>
 
                     </li>
 
                     </li>
 
                 </ul>
 
                 </ul>

Revision as of 10:39, 31 October 2017

P R O T O C O L S

Experiments

Amplification of genes:

  • Synthetically produce a codon-optimised version of yddG.
  • Amplify trpE and aroG from WT E.coli MG1655
  • Point mutations for feedback resistance in trpE and aroG: Using primers with overhangs containing point mutations, and splitting the gene in two, then combining the two parts when inserting in the expression vector.
  • Created vector with combinations of one, two and three genes in the USER casette, using primers with overhangs.

To make the point mutations for trpE and aroG, two sets of vectors for each gene was designed (illustration). Overhangs in the end of the primers enable USER cassette insertion, while the primer overhangs in the center contain a point mutation. When the two parts are being amplified individually, the transformation into vector in expression host will be done with USER ligation.

Vector design

Protein import. USER casette and His tag.
Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.

Expression and production

  • Expression checked with Western blotting: all genes HIS tagged.
  • Tryptophane production by E.coli with one, two or three genes, and nuder different levels of inducing agents evaluated on HPLC

Co-growth of E.coli and yeast

  • Find yeast minimal media where E.coli can grow
  • Grow yeast in same media after E.coli has grown, in order to establish possibility for relationship.
  • Grow tryptophane producing E.coli for different time periods, then remove them and grow auxotrophic yeast in the media containing the produced tryptophane.

Growth of E.coli and yeast in same minimal yeast medium

E. coli strains MG1655 and BL21 were grown in several media in order to find a minimal yeast media where E.coli could survive. With inspiration from (van Summeren-Wesenhagen and Marienhagen, 2014), we decided to grow E.coli in the minimal yeast media YNB with the pH adjusted to 7 instead of 4 as original.
After ON growth of E.coli in YNB pH 7, the media was cleared of E.coli by spinning and filtration, after which it was inoculated with yeast (AM94), to ensure that the E.coli does not produce substances hindering yeast growth (protocol).
This experiment is a prerequisite for our next experiment.

Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli

This experiment utilizes the same protocol as the previous (protocol), but now in YNB pH7 media without a tryptophan source, with tryptophan overproducing E.coli and with a tryptophan auxotrophic yeast strain.This experiment is performed with single, double and triple transformations: That is, E.coli with trpE(fbr), aroG(fbr) and yddG alone or in combinations. The growth of yeast is measured using OD600 measurements to evaluate the successful complementation of the yeast amino acid auxotrophy by E.coli tryptophan production.

Design process



In our design process, we have considered a wide range of possible gene combinations. Genes that when over-expressed would have the greatest impact were chosen. This is due to the time constraints set and simplicity. Initially, we considered simply overexpressing the tryptophane operon, but quickly realised this would be highly downregulated due to negative feedback regulation.

We decided that an exporter would be beneficial by reducing the intracellular Trp concentration, which would release the feedback regulation. We also thought of deleting endogenous trpR, but making such a deletion would make our project overly complicated due to the difficulty of making such a deletion is E.coli.

Find Incell here: