The goal of the Fifth International InterLaboratory Measurement Study is to provide understanding of a standardized process of measuring fluorescence that can be replicated under the same conditions (even under different lab conditions). To accomplish this goal, we, along with other iGEM teams, measured both the Absorbance and GFP fluorescence of bacteria carrying 6 Devices using a plate reader, and compared them to known values.
Absorbance
We tested two methods of standardization. In the first method, our team used varying concentrations of a standard solution of silica beads, modeling E. Coli cells, to create a standard curve. The absorbances of the bacteria cultures were then compared with the standard curve, returning a standard concentration of silica beads that can be compared to other iGEM teams.
In the second method, the absorbance of the bacterial cultures was compared with the colony forming units (CFU) by growing them on plates. In accordance to the iGEM protocol, we counted each bacteria culture on the plates as one CFU. This allows the computation of CFU/abs, which is also a value that can be compared with other iGEM teams.
In the second method, the absorbance of the bacterial cultures was compared with the colony forming units (CFU) by growing them on plates. In accordance to the iGEM protocol, we counted each bacteria culture on the plates as one CFU. This allows the computation of CFU/abs, which is also a value that can be compared with other iGEM teams.
GFP Fluorescence
In order to standardize the GFP fluorescence, we compared the relative strengths of GFP expression of our liquid cultures to a standard fluorescein solution. We then used the concentration of silica beads derived from the absorbance to find the fluorescence per particle, which can be used to determine promoter strength. In theory, the standardized fluorescence per particle unit should be consistent with other iGEM teams.
Reflection
We were able to follow the given procedure and obtain reasonable data.
However, during the Interlab study, we encountered several issues that needed to be addressed so that we could still produce standardized results. We were unable to turn off pathlength correction on our plate reader, so we standardized our measurements by adjusting the gain to be the same for all fluorescence measurements. A constant gain value was set manually to 57 for the raw plate reader measurements. This gain value was based off of the optimal gain value of the fluorescein standard curve.
Another issue was that the bacteria from Device 3 did not appear to fluoresce as much as the other devices, causing us to speculate that there could have been contamination or a misexpressed gene due to mutation. In order to find an answer, we sent the DNA for sequencing. The sequencing result indicated that the transformation was successful and that there were no evident mutation or contamination. Data from last year’s Interlab also suggested that Device 3’s fluorescence should be lower than most other devices.
Overall, we had a lot of fun throughout the experiment and learned a great deal about how difficult it is to make experiments standardized so that any lab can obtain similar results. We are excited to contribute to standardizing a measurement process for fluorescence that can be compared across different labs.
For experimental details and notes about the interlab study, please visit our notebook.
