Summary
The aim of the interlab this year was to assess how close measurements can be when fluorescence is measured all around the world.
Participating in the interlab was a valuable experience, and it allowed us to make use of technology we had at our disposal that otherwise wasn’t relevant to our project. It added an extra dimension to our lab work, and was a lot of fun!
When we first began, it seemed like our tubes were empty as reported by a number of other teams. However, we were successful in resuspending them using TE buffer. After transforming the DNA into E.coli Top10 cells, we followed the plate reader protocol supplied by iGEM HQ using LB broth as our growth medium. This involved first calibrating our plate reader with FITC to make a standard curve, before measuring both fluorescence and absorbance of all of our samples at hourly timepoints over a 6 hour time period.
Results
Figure 1. A standard curve generated by measuring the fluorescence of serial dilutions of FITC stock (uM). There is a linear trend for increased fluorescence as [FITC] increases.
Figure 2. The absorbance of the three test devices and both controls transformed into E.coli and inoculated in LB broth was measured every hour over a 6-hour period. The overall trend shows an increase in absorbance for all samples over time.
Figure 3. The fluorescence of the three test devices and both controls transformed into E.coli and inoculated in LB broth was measured every hour over a 6-hour period. There is a sharp increase in fluorescence after the fifth hour, indicating cell maturity had been reached.
Figure 4. The fluorescence readings for each test device or control was corrected for absorbance for each hour reading. It can be seen that for some devices there are large error bars indicating a lot of variation in the results. In general, the ratio decreased over time for all samples.
Observations
• There were difficulties resuspending the pellets in the tubes received from iGEM HQ, and they had to be rehydrated before use.
• It is hypothesised that the cells will grow better in larger conical flasks compared to falcon tubes, contrary to the protocol suggesting both are equivalent
• The OD600 was measured the morning after the cells were inoculated, then diluted to achieve a certain OD600. However, after making these dilutions again using the Excel file provided by iGEM HQ, it is suggested that the OD600s are checked again before continuing to ensure they are all equal.
• It was found there was great variation in readings when using different settings on the plate reader. Given that iGEM HQ has kept the protocol consistent, it is suggested that the desired plate reader settings are also specified to ensure consistency throughout the results.
• As shown in the results, the data seemed to be quite noisy and clustered around the x-axis, with a sharp increase in readings occurring at the final time point. It is suggested that the starting OD600 is higher, or that the experiment be run for longer to ensure that the complete trend is captured. It is theorised that the data for the first 5 hours is merely noise.
• Taking all measurements on the hour that the samples are collected would allow for ongoing monitoring of the readings, rather than stopping the experiment after 6 hours without knowing if the peak fluorescence reading has been reached.
• It is advised that care is taken when dispensing the solutions from ice into the plate wells, as condensation due to varying temperature can cause condensation on the wells that will interfere with the measurement.
