Difference between revisions of "Team:WLC-Milwaukee/Contribution"

 
(44 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
{{WLC-Milwaukee/Navbar}}
 
{{WLC-Milwaukee/Navbar}}
 
<html>
 
<html>
−
<div id="content_wrapper">
+
<!-- Global site tag (gtag.js) - Google Analytics -->
−
<div class="container-fluid">
+
<script async src="https://www.googletagmanager.com/gtag/js?id=UA-109989683-1"></script>
 +
<script>
 +
  window.dataLayer = window.dataLayer || [];
 +
  function gtag(){dataLayer.push(arguments);}
 +
  gtag('js', new Date());
  
 +
  gtag('config', 'UA-109989683-1');
 +
</script>
 +
<style>
 +
#tolcdata1 {
 +
  max-width: 800px;
 +
}
  
−
<h3>★  ALERT! </h3>
+
#tolcdata3 {
−
<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
+
  max-width: 800px;
−
<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
+
}
 +
 
 +
#tolcdata4 {
 +
  max-width: 400px;
 +
}
 +
 
 +
#tolcdata2 {
 +
  max-width: 450px;
 +
}
 +
 
 +
#tolcdata5 {
 +
  max-width: 800px;
 +
}
 +
 
 +
#tolcdata6 {
 +
  max-width: 800px;
 +
}
 +
 
 +
 
 +
</style>
 +
 
 +
<div id="content_wrapper">
 +
<div class="fluid-container">
 +
<div class="big-header">
 +
<img src="https://static.igem.org/mediawiki/2017/9/91/T--WLC-Milwaukee--Characterization_Header_Lg.jpg">
 +
<h1>Characterization</h1>
 +
</div>
 
</div>
 
</div>
−
<div class="clear"></div>
 
  
  
−
<div class="column full_size">
+
<div class="main">
−
<h1>Contribution</h1>
+
−
<h3>Bronze Medal Criterion #4</h3>
+
−
<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study (to be documented on your InterLab page) and/or improve the characterization of an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2017 part number range. Teams who are working on improving the characterization of an existing part should document their experimental design here, along with an explanation for why they chose that part to improve. Data can also be shown here, but it MUST also be documented on the part's Main Page in the Registry.
+
−
<br><br>
+
−
<b>Special Tracks:</b> Document at least one new substantial contribution to the iGEM community that showcases a project related to BioBricks. This contribution should be central to your project and equivalent in difficulty to making and submitting a BioBrick part.
+
  
−
</p>
+
<h1>Characterization of <a href="http://parts.igem.org/Part:BBa_K1900002:Experience" target="_blank"> BBa_K1900002 </a> (<i>E. coli tolC</i>) </h1>
 +
<p> TolC is a barrel-like trimer protein that spans the outer membrane in <i>E. coli</i>, as well as many other gram-negative bacteria. This protein is of particular interest because of its role in antibiotic resistance. Specifically, TolC is known to form an efflux pump when integrated with AcrA and AcrB. Another interesting function of the protein is bacteriophage infection by the TLS phage. We wanted to test the <i>E. coli</i> TolC functions against other gram-negative bacteria's TolC proteins. To improve on the characterization of the <i>E. coli tolC</i> part, our 2017 iGEM team analyzed functions of the <i>E. coli</i> TolC protein in comparison with other species' TolC proteins expressed in a strain of <i>E. coli</i> lacking the gene. Three assays were done with various antibiotics: minimum inhibitory concentration, zones of inhibition, and TLS phage infection. Key findings include that certain strains' TolC proteins do not function well in <i>E. coli</i>, while others do. This leads to indications surrounding which regions of TolC are necessary for antibiotic efflux, and which may be necessary for bacteriophage infection. With some bioinformatic analysis of the variance in amino acid sequences of the TolC proteins, more precise conclusions can be drawn regarding which exact sites integrate with AcrA and AcrB, as well as the mechanism for bacteriophage infection in the extracellular loops. The results are summarized below. </p>
 +
<br> <br> <br>
 +
<h2> Antibiotic efflux demonstrated by zones of inhibition with Kirby-Bauer assays: </h2>
 +
 
 +
<img id="tolcdata1" class="pic-centered" src="https://static.igem.org/mediawiki/2017/f/f9/T--WLC-Milwaukee--WLC_TolC_data.jpg">
 +
<br> <br> <br>
 +
<h2> Antibiotic efflux capabilities confirmed by minimum inhibitory concentration: </h2>
 +
<br> <br> <br>
 +
<img id="tolcdata3" class="pic-centered"  src="https://static.igem.org/mediawiki/2017/8/83/T--WLC-Milwaukee--WLC_TolC_data3.jpg">
 +
<br> <br> <br>
 +
<h2> Phage infection titer with TLS phage: </h2>
 +
<br> <br> <br>
 +
 
 +
<div class="row">
 +
<div class="col-md-6 pic-col">
 +
<img id="tolcdata4" class="pic-centered" src= "https://static.igem.org/mediawiki/2017/8/8f/T--WLC-Milwaukee--WLC_TolC_data4.jpg">
 
</div>
 
</div>
 +
<div class="col-md-6 pic-col">
 +
<img id="tolcdata2" class="pic-centered" src="https://static.igem.org/mediawiki/2017/c/c6/T--WLC-Milwaukee--WLC_TolC_data2.jpg">
 +
</div>
 +
</div>
 +
<br> <br> <br>
 +
<h2> Bioinformatic Analysis: </h2>
 +
<br> <br> <br>
 +
<img id="tolcdata5" class="pic-centered" src="https://static.igem.org/mediawiki/2017/3/30/T--WLC-Milwaukee--WLC_TolC_data5.jpg">
 +
<br> <br> <br>
 +
<p> The amino acid sequences for regions necessary for AcrA interaction (top) and TLS phage infection (bottom) are shown. Conservation in these sequences translate to the strains' TolC proteins' ability to function in <i>E. coli</i>.
 +
<br> <br> <br>
 +
<img id="tolcdata6" class="pic-centered" src="https://static.igem.org/mediawiki/2017/a/a6/T--WLC-Milwaukee--WLC_TolC_data6.jpg">
  
  
  
  
 +
 +
</div>
  
 
</div>
 
</div>

Latest revision as of 00:07, 21 November 2017

Characterization

Characterization of BBa_K1900002 (E. coli tolC)

TolC is a barrel-like trimer protein that spans the outer membrane in E. coli, as well as many other gram-negative bacteria. This protein is of particular interest because of its role in antibiotic resistance. Specifically, TolC is known to form an efflux pump when integrated with AcrA and AcrB. Another interesting function of the protein is bacteriophage infection by the TLS phage. We wanted to test the E. coli TolC functions against other gram-negative bacteria's TolC proteins. To improve on the characterization of the E. coli tolC part, our 2017 iGEM team analyzed functions of the E. coli TolC protein in comparison with other species' TolC proteins expressed in a strain of E. coli lacking the gene. Three assays were done with various antibiotics: minimum inhibitory concentration, zones of inhibition, and TLS phage infection. Key findings include that certain strains' TolC proteins do not function well in E. coli, while others do. This leads to indications surrounding which regions of TolC are necessary for antibiotic efflux, and which may be necessary for bacteriophage infection. With some bioinformatic analysis of the variance in amino acid sequences of the TolC proteins, more precise conclusions can be drawn regarding which exact sites integrate with AcrA and AcrB, as well as the mechanism for bacteriophage infection in the extracellular loops. The results are summarized below.




Antibiotic efflux demonstrated by zones of inhibition with Kirby-Bauer assays:




Antibiotic efflux capabilities confirmed by minimum inhibitory concentration:







Phage infection titer with TLS phage:







Bioinformatic Analysis:







The amino acid sequences for regions necessary for AcrA interaction (top) and TLS phage infection (bottom) are shown. Conservation in these sequences translate to the strains' TolC proteins' ability to function in E. coli.