Difference between revisions of "Team:Exeter/Design"

 
(60 intermediate revisions by 7 users not shown)
Line 3: Line 3:
  
 
<html>
 
<html>
 +
 +
 +
<script>
 +
 +
$(document).ready(function(){
 +
$('a[href^="#"]').on('click',function (e) {
 +
    e.preventDefault();
 +
 +
    var target = this.hash;
 +
    var $target = $(target);
 +
 +
    $('html, body').stop().animate({
 +
        'scrollTop': $target.offset().top - $('#top_menu_14').height() - $('#top_menu_14').height() - $('#navbar-top').height()
 +
    }, 900, 'swing');
 +
});
 +
});
 +
 +
</script>
 +
 +
  <!-- Row & container for main content of the page -->
 +
<div class="container-fluid">
 +
  <div class="row">
 +
    <div class="col-2">
 +
      <a href="https://2017.igem.org/Team:Exeter"><img id="logo-top-left" width="100%;" src="https://static.igem.org/mediawiki/2017/2/29/T--Exeter--startpage_logo.png"></a>
 +
      <nav id="navbar-page" class="navbar"> <!-- classes removed from template: navbar-light bg-light-->
 +
        <!-- <a class="navbar-brand" href="#navbar-top">Back to top</a> -->
 +
        <nav class="nav nav-pills flex-column">
 +
          <a class="nav-link" href="#h1">Type I Pili</a>
 +
          <a class="nav-link" href="#h2">Plasmid Design</a>
 +
          <a class="nav-link" href="#h3">Real Life Application</a>
 +
          <a class="nav-link" href="#pageHeader">back to top</a>
 +
        </nav>
 +
      </nav>
 +
    </div>
 +
    <div class="col-10" id="pageContent">
 +
      <div>
 +
 +
      <img class="w-25 mx-auto d-block" src="https://static.igem.org/mediawiki/2017/1/18/T--Exeter--Design_logo.png">
 +
        <h1 id="pageHeader">Biological Design</h1>
 +
        <h2 id="h1">Type I Pili</h2>
 
<p>
 
<p>
−
.
+
Pili are small, hair-like structures found on bacterial cells. Pili are encoded by the fim operon which contains a number of genes that code for structural proteins, including FimH. The main constituent is FimA which polymerises to form the main body of the pilus structure and the other proteins are largely related to the chaperone-usher pathway (Busch et. al 2015). FimH is found at the very end of the complex, and contains lectin - the mannose binding domain. They have a natural ability to attach to eukaryotic cell surface mannose molecules due to specific binding sites within FimH, the terminal protein. By investigating adhesion mechanisms involved in type I pili we aimed to repurpose the structures by introducing new metal-binding domains within the pili of our <i>E. coli</i>. 
 +
 
 
</p>
 
</p>
  
−
  <head>
+
<figure class="w-100 mx-auto d-block rounded border border-dark">
−
      <title>Applied Design</title>
+
<img class="w-50 mx-auto d-block" src="https://static.igem.org/mediawiki/2017/7/7d/T--Exeter--pilus.png">
−
      <style>
+
<figcaption style="text-align:center">
−
        @import url('https://fonts.googleapis.com/css?family=Oxygen');
+
<b>Figure 1</b>: Schematic of a type I pilus (adapted from Busch et al 2015)
−
      </style>
+
</figcaption>
−
  </head>
+
</figure>        
−
+
−
<body>
+
−
<font face="oxygen">
+
−
<h1><b><u><center>Applied Design</center></u></b></h1>
+
−
<p>A 2016 report written by Harvard Law School shows that South Africa
+
−
Have failed to adress the adverse environmental and health
+
−
effects of more than 130 years of gold mining in and around
+
−
Johannesburg.</p>
+
−
+
−
<p>Mining, and more specifically the contaminated waste created
+
−
by the mining industry, causes damage to the environment that is
+
−
largely irreversible. Mine waste, or <i>tailings</i>, can contain as many
+
−
as three dozen hazardous chemicals, including lead, arsenic, mercury and cyanide
+
−
to name but a few.</p>
+
−
+
−
<p>Unfortunately, governments in less economically developed countries (LEDCs) are
+
−
not acting quickly enough on what is already a huge and growing
+
−
environmental catastrophe. With the World's leading mining country
+
−
being South Africa, this lack of action taken by the govermnent is
+
−
putting lives at risk.</p>
+
−
+
−
<h2><b>Current Methods</b></h2>
+
−
<p>There are two routes that current treatments can take: active treatment methods require
+
−
energy and chemical usage, whereas passive treatment methods use only natural processes,
+
−
which can include gravity, plants, or even microorganisms.</p>
+
  
−
<p>Such passive processes include Vertical Flow Ponds (VFP’s). The project, which was formed by the University of Newcastle in 2013, was piloted by the Department of Environment, Food and Rural Affairs in partnership with the Coal Authority, the Environment Agency and the National Trust after receiving finding of £1 million to remove heavy metal pollutants leaching from disused mines (Anon., 2013). The scheme bio-remediates water to firstly remove metal contaminants and then allow the outflow to enter wetland where it is filtered through limestone and compost. (Adam Jarvis, 2015).</p>
+
        <h2 id="h2">Plasmid Design</h2>
 +
<p>
 +
We have produced two types of plasmid, using the modular cloning method (Weber, E., et al) that, once transformed into a bacterium, will be able to synthesise the engineered pili capable of binding to metal ions in water. To ascertain the ideal placement of the metal-binding coding sequences, we tested the expression of super-folded GFP at the 1st, 225th and 258th amino acid, where the signal peptide is not included in the residue count. Protein-coding sequences have previously been <a href="http://www.microbiologyresearch.org/docserver/fulltext/micro/141/11/mic-141-11-2839.pdf?expires=1508926376&id=id&accname=guest&checksum=FB8146093A5404C9B5B04AC4B08AADD8">inserted at these three positions</a>, by numerous research groups. Based on our research and results, we decided to insert all the metal-binding proteins at the 1st amino acid following the signal peptide. </p>
  
−
<p>In addition to this there is ongoing research conducted by the GW4 Alliance into using algae as a form of wastewater treatment. Dr Chris Byran and Dr Mark van der Giezen along with researchers from Bristol, Cardiff and Bath Universities as well as Plymouth Marine Laboratories took samples form the Wheal Jane tin mine and attempted culture algae in them. Testing proved that algae was capable of growing in the mine water and showed that the presence of metals resulted into a greater conversion of bio-mass to bio-crude, which is used to make bio-fuels. The research which has attracted the attention of industry and academics alike is hoped to be applied to waste streams in the future.<p>
+
<figure class="border border-dark rounded w-100 mx-auto d-block">
 +
<img class="rounded mx-auto d-block w-75" src="https://static.igem.org/mediawiki/2017/0/07/T--Exeter--scaledplasmids.png">
 +
<figcaption style="text-align:center">
 +
  <b>Figure 2</b>: Example plasmids showing the structure of our constructs
 +
</figcaption>
 +
</figure>
 +
<p>
 +
The first plasmid contains the <i>fimH</i> gene, with an inserted metal-binding protein immediately at the end of the signal peptide <a href="http://www.uniprot.org/uniprot/P08191">at the 1st amino acid</a>. This is intended to minimise issues with the chaperone-usher pathway and open up further opportunities for future FimH modification. Based on the <a href="https://2017.igem.org/Team:Exeter/HP/Fieldtrips">primary water samples we collected from the abandoned Wheal Maid mine</a>, we have chosen to insert four different metal-binding proteins that bind the most present and damaging ions. We have decided to make four different plasmids, with four different metal-binding proteins: </p>
  
−
<p>Active water treatment is far more common. Clarifying agents such as coagulants or flocculants are will later clump together and are then left to settle before they are removed, dried out and eventually disposed of – most commonly in a waste disposal site. This method is dependent on the desired water quality, and sometimes other methods are used such as ion exchangers, membrane filters, and reverse osmosis.</p>
+
<p>The second plasmid contains the remainder of the <i> fim operon </i> containing six <i>fim</i> coding sequences excluding <i>fimH</i> to allow the biosynthesis of the entire pilus structure. This plasmid always needs to be co-transformed with the <i>fimH</i> plasmid that codes for the pertinent fusion protein to produce metal binding pili. This can be used as a reproducible method for modifying the <i>fim operon</i> in pili by producing a new modular toolkit to advance the field of metal extraction. </p>
 +
         
 +
 
 +
        <h2 id="h3">Real Life Application</h2>
 +
<p>We are aware of the high risks associated with involving a genetically modified organism within a real life application. As demonstrated in the research conducted by the <a href="https://2016.igem.org/Team:Exeter">Exeter 2016 iGEM team</a> a kill switch is not a reliable biocontainment method. To take this into account we have developed <a href="https://2017.igem.org/Team:Exeter/Applied_Design">three-stage filtration system</a>,
 +
  consisting of the hydrocyclone, metal binding reactor and a biosecurity mechanism. The GM bacteria will be contained within the metal binding reactor, where the pili will be able to sequester metal ions from water. The water then passes into the biosecurity mechanism, which will kill any bacteria that try to escape.
 +
</p>
  
−
+
<img class="w-25 mx-auto d-block" src="https://static.igem.org/mediawiki/2017/5/53/T--Exeter--logo_filter.png">
−
"Areas affected by contaminated mine water are often damaged for many decades, if not
+
−
centuries. Because the treatment of polluted mine water is usually expensive, active
+
−
treatment is usually used in heavily populated areas, at working mines, or where governmental
+
−
money was made available for treatment purposes." (Wolkersdorfer, C. 2008)
+
  
−
<br><p>However, these methods have proven to have high maintenance and running costs. Hence governments of LEDCs would be unlikely to invest in such technologies when there may be more pressing issues. Additionally the storage of residual waste and its future effect on the environment is becoming a growing concern.</p>
+
          <h2 id="references">References</h2>
 +
          <ul id="referenceList"
 +
            <li id="ref1">Fronzes, R. et al., Architectures and biogenesis of non‐flagellar protein appendages in Gram‐negative bacteria, The EMBO Journal, Volume 27, Issue 17, 2271-2352 (2008)</li>
 +
<li> Busch A., Phan, G., and Waksman, G. (2015) Molecular mechanism of bacterial type 1 and P pili assembly. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 373, 20130153–20130153.
 +
</li>
 +
            <li>Pallesen, L. et al., Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences, Microbiology, 141, 2839-2848 (1995)</li>
 +
<li>Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marrillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One 6, e16765.</li>
 +
                      </ul>
  
−
<p>With this in mind our team at Exeter have spent the summer working on developing a cost effective and sustainable solution which will have a reduced effect on the environment than compared to the existing technologies in use.</p>
+
      </div>
−
<h2><b>Our Solution</b></h2>
+
    </div>
−
<p>The Pili+ Filtration System offers a cheap to run, cheap to produce
+
  </div>
−
alternative to current methods of decontaminating mine waste. It is comprised
+
</div>
−
of three modular components, each of which will be easy to replace or adapt in the
+
−
event of damage or situational change.</p>
+
−
+
−
<p>The first component is the <b><i>hydrocyclone</i></b>.
+
−
The hydrocyclone relies on its conical geometry to filter larger particulates, such
+
−
as sand, from contaminated water. This function is required to prevent blockages in the
+
−
fluidised media reactor, component two. We have chosen to use a hydrocyclone because
+
−
it has no moving parts, requires only a pump, and small versions can easily be
+
−
3D printed, or manufactured using simple and affordable methods. This makes it
+
−
perfect for useage in LEDCs where resources are scarce yet situation is critical.</p>
+
−
+
−
<p>The second component, as mentioned previously, is the <b><i>fluidised media reactor</i></b> (FMR).
+
−
The fluidised media reactor is a piece of cylindrical housing apparatus designed to contain
+
−
our genetically modified bacteria. The water, now free of large particulates, is fed into the top
+
−
of the FMR. It travels down through the central pipe, and slowly rises back through the outer pipe,
+
−
in which the bacteria are contained. The E.Coli are grown on a sponge material, using
+
−
mouthwash which stresses the bacteria and promotes the development of a biofilm. Here,
+
−
we have optimised the flow rateto ensure that the bacteria have a high probability of binding
+
−
to the targeted metal ions.</p>
+
−
+
−
<p>The third component has been designed to ensure the containment of the genetically
+
−
modified bacteria.</p>
+
−
+
−
+
−
<h2><b><u>References (Harvard)</u></b></h2>
+
−
<ol type="A">
+
−
<li>Wolkersdorfer, C., Chapter 11: Mine Water Treatment and Ground Water Protection in Water
+
−
Management at Abandoned Flooded Underground Mines: Fundamentals, Tracer Tests, Modelling, Water
+
−
Treatment, A. International Mine Water and I. ebrary, Editors. 2008, Springer: Berlin. p. 235.</li>  
+
−
</ol>
+
−
</font>
+
−
</body>
+
  
 
</html>
 
</html>
 +
{{Exeter/footer}}

Latest revision as of 03:44, 2 November 2017

Biological Design

Type I Pili

Pili are small, hair-like structures found on bacterial cells. Pili are encoded by the fim operon which contains a number of genes that code for structural proteins, including FimH. The main constituent is FimA which polymerises to form the main body of the pilus structure and the other proteins are largely related to the chaperone-usher pathway (Busch et. al 2015). FimH is found at the very end of the complex, and contains lectin - the mannose binding domain. They have a natural ability to attach to eukaryotic cell surface mannose molecules due to specific binding sites within FimH, the terminal protein. By investigating adhesion mechanisms involved in type I pili we aimed to repurpose the structures by introducing new metal-binding domains within the pili of our E. coli.

Figure 1: Schematic of a type I pilus (adapted from Busch et al 2015)

Plasmid Design

We have produced two types of plasmid, using the modular cloning method (Weber, E., et al) that, once transformed into a bacterium, will be able to synthesise the engineered pili capable of binding to metal ions in water. To ascertain the ideal placement of the metal-binding coding sequences, we tested the expression of super-folded GFP at the 1st, 225th and 258th amino acid, where the signal peptide is not included in the residue count. Protein-coding sequences have previously been inserted at these three positions, by numerous research groups. Based on our research and results, we decided to insert all the metal-binding proteins at the 1st amino acid following the signal peptide.

Figure 2: Example plasmids showing the structure of our constructs

The first plasmid contains the fimH gene, with an inserted metal-binding protein immediately at the end of the signal peptide at the 1st amino acid. This is intended to minimise issues with the chaperone-usher pathway and open up further opportunities for future FimH modification. Based on the primary water samples we collected from the abandoned Wheal Maid mine, we have chosen to insert four different metal-binding proteins that bind the most present and damaging ions. We have decided to make four different plasmids, with four different metal-binding proteins:

The second plasmid contains the remainder of the fim operon containing six fim coding sequences excluding fimH to allow the biosynthesis of the entire pilus structure. This plasmid always needs to be co-transformed with the fimH plasmid that codes for the pertinent fusion protein to produce metal binding pili. This can be used as a reproducible method for modifying the fim operon in pili by producing a new modular toolkit to advance the field of metal extraction.

Real Life Application

We are aware of the high risks associated with involving a genetically modified organism within a real life application. As demonstrated in the research conducted by the Exeter 2016 iGEM team a kill switch is not a reliable biocontainment method. To take this into account we have developed three-stage filtration system, consisting of the hydrocyclone, metal binding reactor and a biosecurity mechanism. The GM bacteria will be contained within the metal binding reactor, where the pili will be able to sequester metal ions from water. The water then passes into the biosecurity mechanism, which will kill any bacteria that try to escape.

References

    Fronzes, R. et al., Architectures and biogenesis of non‐flagellar protein appendages in Gram‐negative bacteria, The EMBO Journal, Volume 27, Issue 17, 2271-2352 (2008)
  • Busch A., Phan, G., and Waksman, G. (2015) Molecular mechanism of bacterial type 1 and P pili assembly. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 373, 20130153–20130153.
  • Pallesen, L. et al., Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences, Microbiology, 141, 2839-2848 (1995)
  • Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marrillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One 6, e16765.