Team:Macquarie Australia/interlab

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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
Macquarie Australia’s iGEM team is very excited to be a part of the 2016 Interlab study [2]. This study builds on previous years attempts to compare results between multiple iGEM teams at an international level.

Aim


Macquarie University iGEM team is participating in the 'Third 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 three test devices that have different promoters and ribosome binding sequences (RBS). This was to calculate expression levels of five different reporter constructs using fluorescence/OD600. At Macquarie, we used a spectrophotometer and 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.

interlab test tubes
The five devices in pSB1C3 plasmid backbone used during the 2016 iGEM interlab study at Macquarie University.



absorbance at 600 nm
The 3 test devices and controls plated out under a fluorescent light.



Methods and Materials



Interlab study protocols.

Interlab Study Protocols.

We transformed five plasmids containing the three constructs (J23101, J23106, J23117) as well as the positive and negative controls (I20270, R0040) into the E. coli DH10 B 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 five plasmids using the plate reader.



Protocol Links

Transformation Protocol: Transformation
LUDOX OD600, FITC Standard Curve and Cell Measurement Protocols:
OD600, FITC and Plate Reader Protocols



Results and Discussion


Neither of the test device 1 replicates showed any signs of growth during the 6 hour growth period after dilution to 0.02 ABS units. This was particularly odd as test device 1 had a similar initial cell density, when compared the other devices and controls prior to dilution. (see figure 2). Test device 2 had the greatest increase in fluorescence compared to test device 3 which had significantly lower fluorescence (figure 1).
Fluorescents of the test devices.
Fig 1. 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.


absorbance at 600 nm
Fig 2. This graph shows the absorbance at 600 nanometres of each cell culture, which
provides an estimate for the number of cells in the samples.


We measured the effect of the test devices on growth. After diluting the initial cell cultures to an absorbance of 0.02, the dilutions (200 uL) were pipetted into 10 wells of a 96 well plate followed by 2 blank samples of chloramphenicol LB (200 uL). This was left overnight at 37⁰C to grow with the OD 600 taken every 6 minutes, the resulting 180 data points were plotted on a graph (figure 3). It showed that the cells transformed with test device 1 experienced a delay in the onset of their log phase by approximately 9 hours. We hypothesise that the promoter for test device 1 could be inhibiting the growth of our organism.

absorbance at 600 nm
Fig 3. Graph of the OD 600 of the liquid cultures of each device and the controls
plotted over 18 hrs.


Conclusions


  • The Macquarie iGEM team were grateful for the opportunity to contribute to the Interlab Study for the first time.
  • As both replicates of test device 1 showed no signs of growth, we undertook our own investigations into the effect of the test device on our organism. We found that the test device 1 caused a lag in the onset of the log phase.
  • We look forward to participating in future Interlab projects.

References


  1. Kwok, R. 2010. Five hard truths for synthetic biology. Nature, 463, 288.
  2. Beal, J., Haddock-Angelli, T., Gershater, M., De Mora, K., Lizarazo, M., Hollenhorst, J. & Rettberg, R. 2016.
    Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PloS one, 11, e0150182.