Difference between revisions of "Team:Exeter/Design"

 
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      <title>Applied Design</title>
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<h1><b><u><center>Applied Design</center></u></b></h1>
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<p>A 2016 report written by Harvard Law School shows that South Africa
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Have failed to adress the adverse environmental and health
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effects of more than 130 years of gold mining in and around
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Johannesburg.</p>
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<p>Mining, and more specifically the contaminated waste created
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by the mining industry, causes damage to the environment that is
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largely irreversible. Mine waste, or <i>tailings</i>, can contain as many
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as three dozen hazardous chemicals, including lead, arsenic, mercury and cyanide
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to name but a few.</p>
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<p>Unfortunately, governments in less economically developed countries (LEDCs) are
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not acting quickly enough on what is already a huge and growing
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environmental catastrophe. With the World's leading mining country
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      <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>
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being South Africa, this lack of action taken by the govermnent is
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putting lives at risk.</p>
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        <!-- <a class="navbar-brand" href="#navbar-top">Back to top</a> -->
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<h2><b>Current Methods</b></h2>
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          <a class="nav-link" href="#h1">Type I Pili</a>
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<p>There are two routes that current treatments can take: active treatment methods require
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          <a class="nav-link" href="#h2">Plasmid Design</a>
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energy and chemical usage, whereas passive treatment methods use only natural processes,
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          <a class="nav-link" href="#h3">Real Life Application</a>
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which can include gravity, plants, or even microorganisms.</p>
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          <a class="nav-link" href="#pageHeader">back to top</a>
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<p>Active water treatment is far more common. Chemicals called coagulants or flocculants are
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added to waste water in order to quickly clump small metal particles together. These clumps
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are then left to settle before they are removed, dried out and eventually disposed of -
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either underground in the mine, or in a waste disposal site. This method is dependent on
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the desired water quality, and sometimes other methods are used such as ion exchangers,
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membrane filters, and reverse osmosis.</p>
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        <h1 id="pageHeader">Biological Design</h1>
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"Areas affected by contaminated mine water are often damaged for many decades, if not
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        <h2 id="h1">Type I Pili</h2>
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centuries. Because the treatment of polluted mine water is usually expensive, active
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treatment is usually used in heavily populated areas, at working mines, or where governmental
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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>. 
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money was made available for treatment purposes." (Wolkersdorfer, C. 2008)
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<p>However, these methods have proven to have high maintenance and running costs. Hence governments
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of LEDCs would be unlikely to invest in such technologies when there may be more pressing issuess.
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Hence, our team at Exeter have spent the Summer working on a cost effective solution.</p>
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<img class="w-50 mx-auto d-block" src="https://static.igem.org/mediawiki/2017/7/7d/T--Exeter--pilus.png">
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<figcaption style="text-align:center">
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<h2><b>Our Solution</b></h2>
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<b>Figure 1</b>: Schematic of a type I pilus (adapted from Busch et al 2015)
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<p>The Pili+ Filtration System offers a cheap to run, cheap to produce
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alternative to current methods of decontaminating mine waste. It is comprised
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of three modular components, each of which will be easy to replace or adapt in the
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event of damage or situational change.</p>
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        <h2 id="h2">Plasmid Design</h2>
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<p>
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<p>The first component is the <b><i>hydrocyclone</i></b>.
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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>
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The hydrocyclone relies on its conical geometry to filter larger particulates, such
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as sand, from contaminated water. This function is required to prevent blockages in the  
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fluidised media reactor, component two. We have chosen to use a hydrocyclone because
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<img class="rounded mx-auto d-block w-75" src="https://static.igem.org/mediawiki/2017/0/07/T--Exeter--scaledplasmids.png">
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it has no moving parts, requires only a pump, and small versions can easily be
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3D printed, or manufactured using simple and affordable methods. This makes it
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  <b>Figure 2</b>: Example plasmids showing the structure of our constructs
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perfect for useage in LEDCs where resources are scarce yet situation is critical.</p>
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<p>The second component, as mentioned previously, is the <b><i>fluidised media reactor</i></b> (FMR).
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The fluidised media reactor is a piece of cylindrical housing apparatus designed to contain
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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>
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our genetically modified bacteria. The water, now free of large particulates, is fed into the top
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of the FMR. It travels down through the central pipe, and slowly rises back through the outer pipe,
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<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>
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in which the bacteria are contained. The E.Coli are grown on a sponge material, using
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mouthwash which stresses the bacteria and promotes the development of a biofilm. Here,
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we have optimised the flow rateto ensure that the bacteria have a high probability of binding
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        <h2 id="h3">Real Life Application</h2>
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to the targeted metal ions.</p>
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<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>,
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  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.
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<p>The third component has been designed to ensure the containment of the genetically
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modified bacteria.</p>
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<h2><b><u>References (Harvard)</u></b></h2>
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          <h2 id="references">References</h2>
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<li>Wolkersdorfer, C., Chapter 11: Mine Water Treatment and Ground Water Protection in Water
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            <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>
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Management at Abandoned Flooded Underground Mines: Fundamentals, Tracer Tests, Modelling, Water
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<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.
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Treatment, A. International Mine Water and I. ebrary, Editors. 2008, Springer: Berlin. p. 235.</li>  
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            <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>
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<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>
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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.