Collaboration with Waterloo

Thus summer, QGEM's Dry Lab created a ratiometric software application. This program works to read user input of designated protein ratio in the biofilm. Based on other user input constraints, such as organism and plasmid backbone, the program then outputs the required promoter and RBS sequences needed to produce such a biofilm.

Given the striking similarity in QGEM's bifunctional biofilm optimization project, to that of Waterloo's prion based, a logical collaboration existed between the two team. This summer QGEM faced a deficit in web development knowledge, which Waterloo was eager to aid with - graciously offering to building a web accessible version of the software for public use. Both teams planned to build upon the existing software program, broadening its bases of organism knowledge. QGEM primarily worked with E.coli this summer, and thus, the program solely contained an E.coli database. In contrast, Waterloo worked with Yeast this summer, and therefore planned to add Yeast data to our organism database. Waterloo hoped to then utilize the program, allowing faster progression of their project - as optimal parts of their organism would be quickly brought to their knowledge, saving them valuable time.

Unfortunately, this collaboration between teams did not happen until late into the summer, and not enough time existed to complete the collaboration to its full extent. Nevertheless, the opportunity to discuss projects to parallel in idea and execution provided imperative problem solving aid to both teams. Waterloo brought to the attention of QGEM, various parameters that should and could be incorporated into the ratiometric program for stronger results. For example, looking into the effects of degradation rates of protein production.

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

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.

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.