Difference between revisions of "Team:Pittsburgh/Demonstrate"

 
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{{Pittsburgh_Header}}
 
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<h2>Fluorescence Results</h2>
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<p>Dronpa is a photoswitchable fluorescent protein that loses fluorescence under 500-nm light and restores fluorescence under 400-nm light. In our fluorescence assays, we wanted to assess if our constructs are expressing Dronpa.</p>
  
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<h3>Plate Reader Fluorescence Assay</h3>
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<p>Since Dronpa fluoresces similarly to (in the range of) GFP, we looked at the fluorescence of each of our strains using the GFP settings of our plate reader. The Dronpa containing constructs fluoresced more than their non-Dronpa counterparts, which confirms expression.</p>
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<p>
 
<p>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve a their gold medals!
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<img src="https://static.igem.org/mediawiki/2017/3/31/T--Pittsburgh--fluorescencepic.jpeg" height="300" width="600">
 
</p>
 
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<h2>Microscopy Results</h2>
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<p>First we looked at controls and non-dronpa constructs to establish a baseline to compare with dronpa. The CheY knockout strain from the Keio collection displayed no swimming regardless of light stimulation, as expected. The CheY overexpression strain displayed tumbling regardless of light.</p>
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<p>Now we could compare the behavior of chey dronpa with our wild type strain. For chey dronpa, we recorded 7 swimmers in the first trial. This is in accordance with the default dimerized state of Dronpa. After 500-nm light, CheY should be reactivated, which agrees with the decrease in swimming we observed in the second trial. In the final trial, we didn't see a resurgence in swimming as we would expect, and we are exploring the possibility that 400-nm light is toxic to E. <i>coli</i>.</p>
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<p>Here we have a table summarizing all of our microscopy results. As you can see, we actually observed many of the behaviors we expected, including critically our Dronpa constructs This indicates that we were successful in controlling E. <i>coli</i> motility using Dronpa.</p>
  
 
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Please see the <a href="https://2017.igem.org/Judging/Medals">2017 Medals Page</a> for more information.
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<img src="https://static.igem.org/mediawiki/2017/f/f7/T--Pittsburgh--microchart.jpeg" height="400" width="600">
 
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<h4> What should we do for our demonstration?</h4>
 
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<h5> Standard teams </h5>
 
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If you have built a proof of concept system, you can demonstrate it working under real world conditions. If you have built a biological device that is intended to be a sensor, can you show it detecting whatever it is intended to sense. If it is intended to work in the field, you can show how this might work using a simulated version in the lab, or a simulation of your device in the field.<strong> Please note biological materials must not be taken out of the lab</strong>.
 
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<h5> Special track teams </h5>
 
  
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<h3>Videos</h3>
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<br>
 
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<p>
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Special track teams can achieve this medal criterion by bringing their work to the Jamboree and showcasing it in the track event. Art & Design, Measurement, Hardware and Software tracks will all have showcase events at the Giant Jamboree.<strong> Please note biological materials must not be taken out of the lab</strong>.
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<h4>In order: 1. Pre-illumination 2. After 2 minutes 500nm light 3. After 3 seconds 400nm light</h4>
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<h4>CheY Knockout</h4>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgbEd5cVV5dzVNVnM/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgWjhKM2paT3lzS00/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgOENWUF9yamw2UWc/preview" width="480" height="360"></iframe>
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<br>
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<br>
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<h4>CheY Wild-type</h4>
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<br>
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<br>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgR2loaWFkS0pGcTQ/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgSG9ndHdDN0xTaVk/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgZDRIWHgxdVVmWnM/preview" width="480" height="360"></iframe>
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<br>
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<br>
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<h4>CheY Wild-type Dronpa Fusion</h4>
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<br>
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<br>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgTXJfNDlDYlFyRm8/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgcmRvVktBbXR5Y2s/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgZ2dsRVV3czZJRU0/preview" width="480" height="360"></iframe>
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<br>
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<br>
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<h4>CheY Mutant</h4>
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<br>
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<br>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgUVRvYXhKOGplSWs/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgclY1OXhLV1d2Tkk/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgdmF2amVJS25sMEE/preview" width="480" height="360"></iframe>
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<br>
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<br>
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<h4>CheY Mutant Dronpa Fusion</h4>
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<br>
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<br>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgZ29CajlwTlVmQmM/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgYnFVZHpWT04zaEk/preview" width="480" height="360"></iframe>
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<iframe src="https://drive.google.com/file/d/0B8DtFUN9dEIgOUp5RzdNSkJ3Znc/preview" width="480" height="360"></iframe>
  
 
</html>
 
</html>

Latest revision as of 23:37, 8 December 2017

Fluorescence Results

Dronpa is a photoswitchable fluorescent protein that loses fluorescence under 500-nm light and restores fluorescence under 400-nm light. In our fluorescence assays, we wanted to assess if our constructs are expressing Dronpa.

Plate Reader Fluorescence Assay

Since Dronpa fluoresces similarly to (in the range of) GFP, we looked at the fluorescence of each of our strains using the GFP settings of our plate reader. The Dronpa containing constructs fluoresced more than their non-Dronpa counterparts, which confirms expression.

Microscopy Results

First we looked at controls and non-dronpa constructs to establish a baseline to compare with dronpa. The CheY knockout strain from the Keio collection displayed no swimming regardless of light stimulation, as expected. The CheY overexpression strain displayed tumbling regardless of light.

Now we could compare the behavior of chey dronpa with our wild type strain. For chey dronpa, we recorded 7 swimmers in the first trial. This is in accordance with the default dimerized state of Dronpa. After 500-nm light, CheY should be reactivated, which agrees with the decrease in swimming we observed in the second trial. In the final trial, we didn't see a resurgence in swimming as we would expect, and we are exploring the possibility that 400-nm light is toxic to E. coli.

Here we have a table summarizing all of our microscopy results. As you can see, we actually observed many of the behaviors we expected, including critically our Dronpa constructs This indicates that we were successful in controlling E. coli motility using Dronpa.


Videos


In order: 1. Pre-illumination 2. After 2 minutes 500nm light 3. After 3 seconds 400nm light


CheY Knockout





CheY Wild-type





CheY Wild-type Dronpa Fusion





CheY Mutant





CheY Mutant Dronpa Fusion