Difference between revisions of "Team:UMaryland/interlab"

 
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<h11>Interlab Study</h11>
 
<h11>Interlab Study</h11>
 
<h21>Fluorescence Data From 3 Different Constructs with GFP</h21>
 
<h21>Fluorescence Data From 3 Different Constructs with GFP</h21>
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<h31>Standardizing fluorescence measurements by adjusting absorbance values and promoting further joint scientific efforts of the iGEM community. </h31>
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<h31>Standardizing fluorescence measurements and promoting further joint scientific efforts of the iGEM community. </h31>
 
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<h41 id="h4-first" >Tested Devices</h41>
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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>All of our devices were constructed in the PSB1C3 backbone. The chassis for all of our devices was E. coli BL21. The positive control was BBa_I20270 (previously characterized promoter::GFP) in PSB1C3 and the negative control was BBa_R0040 (also previously characterized promoter) in PSB1C3</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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<table id="table-devices">
 
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<h51>Device 1: J23101+I13504 in PSB1C3</h51>
 
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<img class="figure" src="img/dev1.png" />
 
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<h51>Device 2: J23106+I13504 in PSB1C3</h51>
 
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<img class="figure" src="img/dev2.png" />
 
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<h51>Device 3: J23117+I13504 in PSB1C3</h51>
 
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<h4>Construction of the Devices</h4>
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<h4>Tested Devices</h4>
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<p>Our team acquired the necessary Biobricks for this interlab study from the distributed iGEM part kits. These Biobricks, each housed in the pSB1C3 vector, were transformed into E. coli K12 DH5-alpha following the standard transformation protocol and incubated overnight on chloramphenicol plates.</p>  
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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>
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<p>Isolated colonies were cultured overnight in LB + chloramphenicol. Clones were isolated using a Qiaprep miniprep kit and subjected to a standard double digest per NEB's protocol for EcoR1-HF and Pst1-HF. The digested plasmids were visualized on a 0.8% agarose gel using a transilluminator to confirm the size of the interlab parts.</p>
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<ul>
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<p>We designed and ordered a series of primers according to NEB's primer design guidelines toward the assembly of the promoter::GFP constructs in the pSB1C3 backbone. In each case, two PCR products would be combined in a Gibson assembly to form the desired plasmid. The pSB1C3 backbone containing each promoter was amplified using the standard forward primer and a promoter-specific reverse primer.</p>  
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<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>
 
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<img class="imageEmbed imgW600" src="img/gfp.png" alt="An image of green fluroescent protein"/>
 
 
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<p>In a second PCR, rbs::GFP (I13504) was amplified with a forward primer that would introduce part of the specific promoter sequence, as well as a standard reverse primer. This allowed for sufficient overlap between the the backbone::promoter and RBS::GFP amplification products for Gibson assemblies.</p>
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<h4>Protocol</h4>
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<p>The PCR cycling conditions and annealing temperatures for each reaction were designed following NEB's standard PCR protocol for Q5 polymerase. The PCR products were purified with Qiagen's PCR clean-up kit and the size of the products verified on an agarose gel. The linear promoter::backbone constructs were then each combined with the amplified I13504 using the Gibson assembly protocol from NEB.</p>  
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<ol>
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<p>The Gibson products were then transformed into BL21 and the constructs were isolated and insert size confirmed as per above, with additional verification through gene sequencing by Genewiz. However, our team was unable to assemble the third device of the J23117 promoter and GFP. Thankfully, this DNA construct was graciously donated to us with iGEM approval from the William and Mary 2015 iGEM team. The controls for this experiment were transformed directly onto BL21. The plasmids were miniprepped and verified through gene sequencing as well.</p>
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<li>Pick two colonies from each plate to inoculate and grow overnight.
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<h4>Testing the Devices</h4>
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</li>
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<p>Our team followed the 2015 InterLab protocol for testing that was posted by iGEM for this study. Three separate biological replicates for the verified constructs and controls were grown in 10 mL culture tubes with loose caps in an incubator overnight for 16-18 hours. We recorded the OD500 of the cells and the samples were diluted to within 5% of 0.5. 150 microliters of three biological replicates of each diluted sample and three technical replicates were transferred to a 96 well plate using a p-200 pipette.</p>  
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<li>Calibrate the plate reader-
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<p>The fluorescence of the samples was then quantified using the SpectraMax M2 microplate reader, at an excitation wavelength of 498 nm and an emission recorded at 541 nm with the auto cutoff setting on. We are also very thankful to Dr. Bentley's lab on campus for allowing us to use their plate reader for testing. The results of the experiments were processed are reported below in arbitrary units.</p>
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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>
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<li>Use FITC to create a standard fluorescence curve -
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Measure fluorescence of FITC at different concentrations to generate a curve of fluorescence over concentration.</li>
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<li>Measure OD600  of the overnight cultures and create dilutions to put them at an OD600 of 0.02.</li>
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<li>Incubate at 37 C and take 100ul samples every hour (from hours 0-6).</li>
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<li>Measure OD600 of all the samples and record.</li>
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</ol>
 
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<h41>Results</h41>
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<h4>Results</h4>
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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>
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<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 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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Latest revision as of 13:37, 28 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.