Difference between revisions of "Team:UAlberta/Description"

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                  <a class="dropdown-item" href="https://2018.igem.org/Team:UAlberta/Description">Description</a>
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                  <a class="dropdown-item" href="https://2018.igem.org/Team:UAlberta/Design">Design</a>
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                  <a class="dropdown-item" href="https://2018.igem.org/Team:UAlberta/Experiments">Experiments</a>
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      <img src="https://static.igem.org/mediawiki/2018/6/68/T--UAlberta--HoveApiaries.png" class="img-fluid" alt="...">
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        <h2>Overview</h2>
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        <p>To make biology easier to engineer, standardization and other engineering principles have been adapted for and applied
 +
        to biological parts. Though, standardization must not only be implemented to parts themselves but also in standardizing
 +
        the methods we use to test these parts so that direct comparisons of designs are possible.</p>
 +
        <div class="row">
 +
          <div class="col-lg-6">
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            <p>Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response
 +
            of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent
 +
            proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be
 +
            linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard
 +
            protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing
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            fluorescence measurements between instruments and different instances of measurement.</p>
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          </div>
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          <div class="col-lg-6 align-self-center">
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            <figure class="figure">
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              <img src="https://static.igem.org/mediawiki/2018/6/68/T--UAlberta--HoveApiaries.png" class="figure-img img-fluid rounded" alt="...">
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              <figcaption class="figure-caption text-right">A caption for the above image.</figcaption>
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            </figure>
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          </div>
 +
        </div>
 +
        <p>Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing fluorescence measurements between instruments and different instances of measurement.</p>
 +
        <div class="row">
 +
          <div class="col-lg-6 align-self-center">
 +
            <figure class="figure">
 +
              <img src="https://static.igem.org/mediawiki/2018/6/68/T--UAlberta--HoveApiaries.png" class="figure-img img-fluid rounded" alt="...">
 +
              <figcaption class="figure-caption">A caption for the above image.</figcaption>
 +
            </figure>
 +
          </div>
 +
          <div class="col-lg-6">
 +
            <p>Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response
 +
            of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent
 +
            proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be
 +
            linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard
 +
            protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing
 +
            fluorescence measurements between instruments and different instances of measurement.</p>
 +
          </div>
 +
        </div>
 +
        <p>For the 2018 InterLab Study, the variability in fluorescence measurements when assessing a population of cells was
 +
        investigated and the utility of normalizing measurements to absolute cell count or colony forming units (CFU) was
 +
        assessed <a href="">[1]</a>. Team UAlberta accomplished these objectives this by following the InterLab Study protocols to measure the
 +
        fluorescence and cellular density of eight devices which were calibrated against established standards.</p>
 +
        <h2>Materials and Methods</h2>
 +
        <p>The calibration and measurement procedures were performed by the members of Team UAlberta as outlined in the 2018
 +
        InterLab Study Protocols. To view the 2018 InterLab Study Protocols, click <a href="">here</a>. The following protocols describe Team
 +
        UAlberta’s methods when none was specified by the InterLab protocols and to provide information about the measurement
 +
        instruments.</p>
 +
      </div>
 +
    </div>
  
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<h1>Project Description</h1>
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<h1>Combating <em>N. ceranae</em> infections in honey bees with porphyrin</h1>
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    <h2><em>Nosema ceranae</em>, the fungal freeloader</h2>
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      <p>
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        <em>Nosema ceranae</em>, a fungus which parasitizes bees, has recently been detected in the major commercial honey bee species, <em>Apis mellifera</em> (European honeybee). <em>N. ceranae</em> invades epithelial cells in the bee midgut, resulting in the debilitating nosemosis disease. Like all microsporidian fungi, <em>N. ceranae</em> lack mitochondria making them dependent on its honey bee host for its energy source. Thus, <em>Nosema</em> infections results in energetic stress and has been implicated in reduced longevity, immune function, and performance of commercial honey bees, causing decreased hive productivity. Due to the integral role that bees have in agriculture and in the environment, the adverse effects of <em>N. ceranae</em> to honey bees inspires much anxiety among the apiculture community.
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    <h2>Fumagilin: Treatment to bees, or not to be?</h2>
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        Team UAlberta was alerted of the <em>N. ceranae</em> threat by our discussions with local beekeepers and researchers, who expressed a desire for effective treatments against <em>Nosema</em> infections. Current methods of treating <em>N. ceranae</em> infections rely heavily on  an antifungal agent called Fumagilin-B.  However, Fumagilin-B is mutagenic, toxic to mammals, and has been shown to increase <em>N. ceranae</em> spore count at low doses. Moreover, the only North American supplier of Fumagilin-B has gone out of business and the remaining supply of Fumagilin-B is beginning to run low. Our discussions with the beekeeping community in Alberta, Canada revealed tremendous concern about the lack of an alternative safeguard against <em>N. ceranae</em>.
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        The honey industry's impact on the Canadian economy also drives much of the concern regarding <em>Nosema</em>. Between honey production and the contribution of their pollination to agriculture, honey bees contributed roughly $2 Billion to the Canadian economy in 2013. The economic impact of the bee industry in Canada is far-reaching and a lack of fumagillin alternatives poses a serious threat to industry and individual livelihoods. The developments regarding the supply of Fumagilin-B and the effects of nosemosis on hive productivity motivates Team UAlberta to develop an alternative treatment against <em>Nosema</em>.
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    <h2>Porphyrins</h2>
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      <p>
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        Recent research has found that porphyrins, a class of organic compounds, are capable of deactivating <em>N. ceranae</em> spores. Porphyrin antifungal action is attributed to its disruption of spore cell walls (Figure 1). When bee diets were supplemented with chemically synthesized porphyrin species, spore count in the bee's midgut significantly decreased while no adverse effects on the bees were observed. Particularly, a porphyrin derivative, PP(Asp)2 was successful in reducing the spore load in treated bees. Therefore, using porphyrin-type molecules like PP(Asp)2 may be a feasible method of treating <em>N. ceranae</em> infections.
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      </p>
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    <h2>Plan Bee</h2>
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        Luckily, PP(Asp)2 is structurally similar to protoporphyrin IX, a porphyrin-type molecule produced endogenously in <em>E. coli</em>. Rather than feeding bees chemically synthesized PP(Asp)2, we aim to genetically engineer a strain of <em>E. coli</em> capable of living in the bee midgut to biosynthesize protoporphyrin IX. We will build off of the work of a previous iGEM team (BeeColi; NYMU-Taipei , 2013) that developed an alginate coat to allow <em>E. coli</em> to travel through the harsh environment of the bee stomach to colonize the bee midgut. Once in the midgut, our <em>E. coli</em> will secrete excess porphyrin IX using an endogenous efflux pump (TolC). Given that ingested porphyrin IX has been shown to damage <em>N. ceranae</em>, we hypothesize that porphyrin IX secreted directly into the midgut will also damage <em>N. ceranae</em>, allowing us to "bee" part of the <em>Nosema</em> solution.
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      </p>
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      <h3>Objective One: <em>Engineer constructs exploiting C5 heme biosynthesis pathway to selectively express and secrete porphyrin intermediates</em></h3>
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      <img src="https://static.igem.org/mediawiki/2018/3/37/T--UAlberta--PorphyrinPathway.png" alt="Proposed porphyrin pathway"></img>
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      <p>
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        <em>Figure 1: Proposed process for the production of protoporphyrin IX by means of engineered E. coli through exploiting the C5 pathway of heme synthesis.</em>
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      </p>
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      <p>
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        First, a liquid chromatography mass spectroscopy assay will be conducted to analyze the distribution of porphyrin-type species produced in wild-type <em>E. coli</em>, establishing a baseline for manipulation of heme biosynthesis. Next, strains of <em>E. coli</em> will be engineered to produce target porphyrin intermediates in the C5 pathway with high yield, by overexpressing genes encoding heme biosynthesis enzymes (Figure 4). To facilitate the activity of porphyrins in the bee midgut, and to avoid possible complications arising from negative feedback loops, we will engineer the <em>E. coli</em> to constitutively secrete the produced porphyrins.  Previous work has shown that the outer membrane channel-tunnel protein, TolC, functions with efflux pumps to export excess porphyrins and maintain homeostasis. Thus, with control over porphyrin intermediate accumulation, secretion of the compounds is achievable through existing cellular machinery. It should be noted that an introduction of a kill switch may be needed to prevent the unwanted proliferation of modified <em>E. coli</em>.
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      </p>
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      <h3>Objective Two: <em>Assay modified strains and associated porphyrin intermediates for their ability to inactivate N. ceranae spores in bees.</em></h3>
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      <p>
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        This assay will be performed using spores from an in vivo source, <em>A. mellifera</em> worker bees, and an in vitro source, Sf9 cells which are permissive to N. ceranae infection. Spores will be incubated with the engineered <em>E. coli</em> and infectivity will be quantified, using conventional PCR and fluorescent cell staining, which are consistent with established methods. A previous iGEM team (NYMU-Taipei 2013) has shown that modified <em>E. coli</em> are capable of persisting within the gut of bees. A similar protocol will be employed to introduce the <em>E. coli</em> into live bees and  the capability of the <em>E. coli</em> to confer extended resistance against <em>N. ceranae</em> will be measured by spore count. The total load of <em>E. coli</em> will be observed to evaluate the response of the microbiome to the modified strain.
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Revision as of 00:14, 17 October 2018

...

Overview

To make biology easier to engineer, standardization and other engineering principles have been adapted for and applied to biological parts. Though, standardization must not only be implemented to parts themselves but also in standardizing the methods we use to test these parts so that direct comparisons of designs are possible.

Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing fluorescence measurements between instruments and different instances of measurement.

...
A caption for the above image.

Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing fluorescence measurements between instruments and different instances of measurement.

...
A caption for the above image.

Thus, the goal of iGEM InterLab studies is to develop standards and protocols for measuring the fluorescence response of fluorescent proteins, a quantity commonly used in evaluating the performance of biological systems. Fluorescent proteins, such as Green Fluorescent Protein (GFP), have become widespread tools as their fluorescence readout can be linked with parameters like gene expression and protein interactions. Although, the lack of a widely accepted standard protocol and variance in measurement instruments, like microplate readers, contributes to the difficulty in comparing fluorescence measurements between instruments and different instances of measurement.

For the 2018 InterLab Study, the variability in fluorescence measurements when assessing a population of cells was investigated and the utility of normalizing measurements to absolute cell count or colony forming units (CFU) was assessed [1]. Team UAlberta accomplished these objectives this by following the InterLab Study protocols to measure the fluorescence and cellular density of eight devices which were calibrated against established standards.

Materials and Methods

The calibration and measurement procedures were performed by the members of Team UAlberta as outlined in the 2018 InterLab Study Protocols. To view the 2018 InterLab Study Protocols, click here. The following protocols describe Team UAlberta’s methods when none was specified by the InterLab protocols and to provide information about the measurement instruments.