Difference between revisions of "Team:UMaryland/Measurement"

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Revision as of 01:30, 10 October 2016

</div> </div> Human Practices

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 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.