Team:Queens Canada/Collaborations

Queen's Canada iGEM team was very excited to collaborate with various other iGEM teams this past summer. Together, we were able to build a stronger iGEM community, and progress in our respective projects - the insight of others providing great advancement.


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



This year, QGEM collaborated with the Ontario Genetically Engineered Machine Collective, which is composed of iGEM teams from the universities of Toronto, Guelph (new this year), Waterloo, McMaster, and Ottawa. With oGEM, we meet several times during the year to discuss ways to promote iGEM in Ontario, funding strategies, project troubleshooting, and new ways to collaborate for wet lab, dry lab, and policy and practice departments.

One of the oGEM collaborations featured this year was promoting iGEM to new potential teams, and creating a detailed database of helpful tips and tricks for wiki development - a task most teams noticeably struggle with. Through web conferencing, email, and simply communicating ideas in person at the oGEM meetings, these valuable tidbits and thoughts were shared. The ability to discuss QGEM's wiki struggles with others, and gain insight into possible methods of remediation, was extremely valuable to QGEM.


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.