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Revision as of 22:41, 11 August 2017


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
    •

Queen's Canada iGEM team is very excited to be a part of the 2017 Interlab study. This study builds on previous years attempts to compare results between multiple iGEM teams at an international level.


Aim

Queen's University iGEM team is participating in the 'Fourth International Interlaboratory Measurement Study' for the first time, which aims to better understand the measuring of fluorescence using a standardised protocol to quantify the variability across different laboratories.

Participating iGEM teams measured fluorescence exhibited from the green fluorescent protein (GFP) across six test devices that have different ribosome binding sequences (RBS). A positive and negative control are also used to calculate expression levels using fluorescence/OD600. At Queen's, we used a plate reader to measure fluorescence, following standard iGEM protocols. We hope that in the future the scientific community can better compare and communicate results with each other. This collaborative effort is a small but meaningful step towards that goal.




Methods and Materials



Interlab study protocols.


Interlab Study Protocols.

We transformed six plasmids containing the three constructs (J23101, J23106, J23117) as well as the positive and negative controls into the E. coli DH5a strain. After growing the cells, we started the calibration protocols of OD600 reference point using the LUDOX solution, FITC as the standard for fluorescence and the cell measurements of eight plasmids using the plate reader. We measured fluorescence at an excitation wavelength 395nm and emission wavelength of 508nm [2].



Protocol Link

Click Here: Interlab Protocol



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 change in fluorescence of each test device over a 6 hour
incubation period. Test device 2 had the greatest increase in fluorescence.

Cultures were sampled at the += 0,2,4,6 hour marks in 500ml aliquots from 10ml cultures. Test device 1 had a highly stagnant growth. Mady won't see this hurr durr.


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.

All samples were added to a 96-well plate to measure fluorescence intensity. Fluorescent values were normalized following measurement.


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 detected by our plate reader, and this threshold is only reached when bacterial OD reaches approximately 0.2 OD on our normalized 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.