Difference between revisions of "Team:UMaryland/Measurement"

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{{UMaryland}}
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<html lang="en">
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<title>Interlab Study</title>
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<ul>
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<a style="opacity: 0">Fix</a>
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<li><a href="https://2016.igem.org/Team:UMaryland/projects">Projects</a></li>
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<!--<li><a href="https://2016.igem.org/Team:UMaryland/parts">Parts</a></li>
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<li><a href="https://2016.igem.org/Team:UMaryland/modeling">Modeling</a></li>-->
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<li><a href="https://2016.igem.org/Team:UMaryland/outreach">Outreach</a></li>
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<li><a href="https://2016.igem.org/Team:UMaryland/notebook">Notebook</a></li>
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<li><a href="https://2016.igem.org/Team:UMaryland/about">About Us</a></li>
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<li><a href="https://2016.igem.org/Team:UMaryland/notebook">Notebook</a></li>
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<h11>Interlab Study</h11>
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<h21>Fluorescence Data From 3 Different Constructs with GFP</h21>
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<h31>Standardizing fluorescence measurements and promoting further joint scientific efforts of the iGEM community. </h31>
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</div>
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<!-- Heading text -->
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<div class="longText">
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<p>The interlab study measures the fluorescence of three different constructs with GFP as well as a positive and negative control. It attempts to standardize the fluorescence measurements by adjusting absorbance values when compared to blanks, reducing the amount of independent variables by controlling for concentration, and adjusting fluorescence values based on the standard curve of FITC.  The goal of this study is to see how the observed fluorescence values compare across the iGEM community to see if the adjustments successfully standardize fluorescence measurements.</p>
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<p>As a community of synthetic biologist, a key to the advancement of the field is collaboration among groups. For proper collaboration, we must be able to communicate our findings in ways that are concrete and well defined, making standards essential.</p>
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</div>
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<div class="longText">
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<h4>Tested Devices</h4>
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<p>Transform competent cells with Test Devices 1 - 3, positive control, and negative control provided and plate them overnight on chloramphenicol resistant plates.</p>
 +
<ul>
 +
<li>Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3</li>
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<li>Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3</li>
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<li>Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3</li>
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<li>Positive Control Device: I20270 in pSB1C3</li>
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<li>Negative Control Device: R0040 in pSB1C3</li>
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</ul>
 +
</div>
 +
<div class="longText">
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<h4>Protocol</h4>
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<ol>
 +
<li>Pick two colonies from each plate to inoculate and grow overnight.
 +
</li>
 +
<li>Calibrate the plate reader-
 +
Measure absorbance of LUDOX and water to find a corrected absorbance that can then be used to find a conversion factor for a standard OD600 measurement.</li>
 +
<li>Use FITC to create a standard fluorescence curve -
 +
Measure fluorescence of FITC at different concentrations to generate a curve of fluorescence over concentration.</li>
 +
<li>Measure OD600  of the overnight cultures and create dilutions to put them at an OD600 of 0.02.</li>
 +
<li>Incubate at 37 C and take 100ul samples every hour (from hours 0-6).</li>
 +
<li>Measure OD600 of all the samples and record.</li>
 +
</ol>
 +
</div>
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<div class="longText">
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<h4>Results</h4>
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<div id="div-results">
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<ul>
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<li>Most samples grew exponentially, device 1 replicate 1 and device 1 replicate 2 seemed to have little growth.</li>
 +
<li>Negative control and device 3 did not show fluorescence. Positive control, device 1, and device 2 showed increases in fluorescence over time.</li>
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<li>The results show that fluorescence per unit of concentration remains relatively constant over time.</li>
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</ul>
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</div>
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<div id="div-figures">
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<img class="figure" src="https://static.igem.org/mediawiki/2016/a/ae/T--UMaryland--absorbance.JPG">
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<small class="caption">Figure 1. Cell growth versus absorbance.</small>
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<img class="figure" src="https://static.igem.org/mediawiki/2016/1/17/T--UMaryland--fluorescence.JPG">
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<small class="caption">Figure 2. Cell growth versus flourescence.</small>
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<img class="figure" src="https://static.igem.org/mediawiki/2016/4/43/T--UMaryland--abs-fluor.JPG">
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<small class="caption">Figure 3. Cell growth versus absorbance / flourescence.</small>
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</div>
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the<a href="https://2016.igem.org/Judging/Awards"> Best Measurement award</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<p>There are a lot of exciting Parts in the Registry, but many Parts have still not been characterized. Synthetic Biology needs great measurement approaches for characterizing new parts, and efficient new methods for characterizing many parts at once. If you've done something exciting in the area of Measurement, describe it here!</p>
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<h5>Inspiration</h5>
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        td {
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<p>You can look at what other teams did to get some inspiration! <br />
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            vertical-align: top;
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Here are a few examples:</p>
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        }
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<ul>
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<li><a href="https://2014.igem.org/Team:Aachen">2014 Aachen  </a></li>
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<li><a href="https://2014.igem.org/Team:Valencia_Biocampus">2014 Valencia Biocampus</a></li>
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<li><a href="https://2015.igem.org/Team:William_and_Mary">2015 William and Mary</a></li>
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</ul>
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                <a href="mailto:umarylandigem@gmail.com"><p id="contact"> Contact Us </p></a>
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            <p id="copyright">© University of Maryland iGEM 2016</p>
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Revision as of 22:15, 26 September 2016

Interlab Study

Interlab Study Fluorescence Data From 3 Different Constructs with GFP Standardizing fluorescence measurements and promoting further joint scientific efforts of the iGEM community.

The interlab study measures the fluorescence of three different constructs with GFP as well as a positive and negative control. It attempts to standardize the fluorescence measurements by adjusting absorbance values when compared to blanks, reducing the amount of independent variables by controlling for concentration, and adjusting fluorescence values based on the standard curve of FITC. The goal of this study is to see how the observed fluorescence values compare across the iGEM community to see if the adjustments successfully standardize fluorescence measurements.

As a community of synthetic biologist, a key to the advancement of the field is collaboration among groups. For proper collaboration, we must be able to communicate our findings in ways that are concrete and well defined, making standards essential.

Tested Devices

Transform competent cells with Test Devices 1 - 3, positive control, and negative control provided and plate them overnight on chloramphenicol resistant plates.

  • Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
  • Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
  • Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
  • Positive Control Device: I20270 in pSB1C3
  • Negative Control Device: R0040 in pSB1C3

Protocol

  1. Pick two colonies from each plate to inoculate and grow overnight.
  2. Calibrate the plate reader- Measure absorbance of LUDOX and water to find a corrected absorbance that can then be used to find a conversion factor for a standard OD600 measurement.
  3. Use FITC to create a standard fluorescence curve - Measure fluorescence of FITC at different concentrations to generate a curve of fluorescence over concentration.
  4. Measure OD600 of the overnight cultures and create dilutions to put them at an OD600 of 0.02.
  5. Incubate at 37 C and take 100ul samples every hour (from hours 0-6).
  6. Measure OD600 of all the samples and record.

Results

  • Most samples grew exponentially, device 1 replicate 1 and device 1 replicate 2 seemed to have little growth.
  • Negative control and device 3 did not show fluorescence. Positive control, device 1, and device 2 showed increases in fluorescence over time.
  • The results show that fluorescence per unit of concentration remains relatively constant over time.
Figure 1. Cell growth versus absorbance. Figure 2. Cell growth versus flourescence. Figure 3. Cell growth versus absorbance / flourescence.