Team:Queens Canada/InterLab

Queen's Canada iGEM team is very excited to be a part of the 2017 Interlab study. This study builds upon previous years attempts to answer one major question:

How close can the numbers be when fluorescence is measured all around the world?


Agar plate streaked with negative control device (left) and positive control device (right) viewed under UV light. Looks like they work!



In 2017, the Queen's University iGEM team is participating in the Fourth International Interlaboratory Measurement Study for their first time. The Interlab Study aims to analyze and improve the replicability of fluorescence measurements. This year, the reliability of some RBS devices (BCDs) will be tested all around the world, using the expression of green fluorescent protein (GFP) to quantify translation.

Participating iGEM teams measure fluorescence exhibited by the GFP across six test devices, each with different RBS sequences. A positive and negative control are also used to calculate expression levels using fluorescence/OD600. Reproducibility is one of the most challenging and critical aspects of scientific research. We hope that the data from this study can help establish a baseline for GFP measurement reproducibility, given GFP's widespread use as a tool in molecular biology. This collaborative effort is a small but meaningful step towards this goal.


Background



The ability to reproduce results in biological systems is difficult due to the stochastic nature of living cells and inconsistent laboratory practices [1]. Comparing quantitative results between experiments is often difficult with many variables impacting the results. These may include:

  • The various instruments used and their different calibrations
  • Variation in laboratory practices/protocols
  • Systematic variability e.g. differences in strains used, physical laboratory conditions
  • Variation in interpreting and communicating results

Methods and Materials




Interlab study protocols.
Interlab Study Protocols

E. coli DH5a strain. After picking two colonies from each transformation, we grew up the cells and started the calibration protocols of OD600 reference point using the LUDOX solution. FITC was used as the standard for fluorescence. We used a Molecular Devices SpectraMax M2e plate reader on the topreading setting for both our OD600 and fluorescent measurements. Black 96-well plates with clear bottoms were used. We measured fluorescence at an excitation wavelength 395nm and emission wavelength of 508nm [2].



Interlab Protocols (PDF)

Results and Discussion



absorbance at 600 nm
Fig 1. Fluorescein standard curve.

The above fluorescence calibration curve (Fig. 1) was created by measuring fluorescence intensity of different concentrations of fluorescein.

Fluorescents of the test devices.
Fig 2. This graph shows the fluorescence of each test device measured every two hours over a span of 6 hours.

Cultures were sampled at the += 0,2,4,6 hour marks in 500ml aliquots from 10ml cultures. All samples were added to a 96-well plate to measure fluorescence intensity. Fluorescent values were normalized prior to plotting. All points on the graphs are the average of the two colonies grown (which themselves are the average of 4 wells each). Test device 2 had the greatest overall increase in fluorescence. Both the negative control device and the LB+ chloramphenicol sample had no significant increase in fluorescence.


absorbance at 600 nm
Fig 3. This graph shows the absorbance at 600 nanometres of each cell culture, which
provides an estimate for the number of cells in the samples.

Every test device exhibits steady, somewhat sigmoidal bacterial growth. The LB + chloramphenicol sample shows no change in OD600 over time.

Conclusions



  • The Queen's_Canada iGEM team was grateful for the opportunity to contribute to the Interlab Study for the first time.
  • It appears that our cells only began expressing significant amounts of GFP after the 4-hour mark. One would expect the curve of increasing GFP fluorescence to mirror the curve of OD600, if GFP expression is truly constitutive. The OD600 curve shows steady, somewhat sigmoidal growth, while the fluorescent intensity curve is a plateau until after 4 hours have elapsed.
  • This suggests either a certain threshold concentration of GFP is required to be detectable by our plate reader, or that GFP expression only begins at a certain cell density threshold (which is reached at approximately 0.2 OD on our Figure 3 graph).
  • Both the LB + chloramphenicol and negative control wells showed no significant increase in fluorescence, as expected.

References



  1. Kwok, R. 2010. Five hard truths for synthetic biology. Nature, 463, 288.
  2. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. 1994. Green Fluorescent Protein as a Marker for Gene Expression. Science: 263(5148), 802-805.



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