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| − | <h1>Overview</h1> | + | <h1>Demonstrate</h1> |
| − | <div class="ua-collapsable">
| + | <p>Over the course of this iGEM season, with parallel efforts in both our bee lab and synbio lab to maximize our limited time, Team UAlberta was able to perform key experiments that together serve as a proof-of-principle demonstration that our system works, which we will recap here. For full experimental results, please proceed to the results section for the bee and synbio lab</p> |
| − | <div class="ua-collapse-button" data-toggle="collapse" data-target="#collapseExample" aria-expanded="false"
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| + | <p>Our solution to addressing <i>N. ceranae</i> infection is predicated on two hypotheses:</p> |
| − | <h6>Press Me</h6>
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| − | <i class="fas fa-arrow-down"></i>
| + | <ol> |
| − | </div>
| + | <li> |
| − | <div class="collapse ua-collapse-info" id="collapseExample">
| + | We can engineer <i>E. coli</i> to overexpress the enzymes needed to drive the production of PPIX.</li> |
| − | <div class="card card-body">
| + | <li>PPIX is effective in reducing the spore load of <i>N. ceranae</i></li> |
| − | Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. Nihil anim keffiyeh
| + | </ol> |
| − | helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident.
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| − | </div>
| + | <h2>Engineering <i>E. coli</i> to overproduce PPIX production<h2> |
| − | </div>
| + | |
| − | </div>
| + | <p>To address the first hypothesis, we performed two experiments. Our first experiment involved overexpressing 5-aminolevulinic synthase, which has been previously shown to be sufficient to drive PPIX production, in <i>DH10B</i> to see if PPIX can be produced as the literature suggests [1]. We extracted porphyrins secreted in the media and characterized them via fluorescence spectroscopy and thin layer chromatography. The similarities of the fluorescence and TLC results between our stock (Enzo Life Sciences) and extracted porphyrins strongly suggest that overexpressing the enzyme encoded by HemA alone is sufficient to drive porphyrin production - specifically PPIX production.</p> |
| − | <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-7">
| + | |
| − | <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 class="col-lg-5 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 text-right">A caption for the above image.</figcaption>
| + | |
| − | </figure>
| + | |
| − | </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">
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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="...">
| + | |
| − | <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> |
| | </div> | | </div> |
Demonstrate
Over the course of this iGEM season, with parallel efforts in both our bee lab and synbio lab to maximize our limited time, Team UAlberta was able to perform key experiments that together serve as a proof-of-principle demonstration that our system works, which we will recap here. For full experimental results, please proceed to the results section for the bee and synbio lab
Our solution to addressing N. ceranae infection is predicated on two hypotheses:
-
We can engineer E. coli to overexpress the enzymes needed to drive the production of PPIX.
- PPIX is effective in reducing the spore load of N. ceranae
Engineering E. coli to overproduce PPIX production
To address the first hypothesis, we performed two experiments. Our first experiment involved overexpressing 5-aminolevulinic synthase, which has been previously shown to be sufficient to drive PPIX production, in DH10B to see if PPIX can be produced as the literature suggests [1]. We extracted porphyrins secreted in the media and characterized them via fluorescence spectroscopy and thin layer chromatography. The similarities of the fluorescence and TLC results between our stock (Enzo Life Sciences) and extracted porphyrins strongly suggest that overexpressing the enzyme encoded by HemA alone is sufficient to drive porphyrin production - specifically PPIX production.
