In addition to the experiments that were conducted to test our project, our team also participated in the iGEM Interlab Study. The goal of this study is to obtain large amounts of data from labs across the world to help develop a more reliable measurement procedure for green fluorescence protein. Instead of getting relative measurements for intensity, the goal of the Interlab Study is to develop absolute units for green fluorescence in a plate reader to eliminate variation between labs.
In order to obtain a more objective calibration for later data, standard OD600 measurements were taken without any sort of pathlength correction to eliminate instrument dependence. LUDOX solution was used to obtain a radiometric conversion factor for later experiments. Our calibration results are listed below in Table 1:
Fluorescein Standard Curve
The next step in the procedure was to create a calibration curve for the fluorescein concentrations. Serial dilutions of the protein were prepared. Four replicates were made for each dilution series in order to get a more reliable average. Both the linear and logarithmic calibration curves are shown below in Figures 1 and 2, respectively:
Once again, pathlength correction was not active for this portion of the experiment, to minimize the effect of instrument error on the measurements. Gain was adjusted in order to eliminate oversaturation.
In order to perform the cell measurement component of the interlab study, 8 plasmids were transformed into E. coli DH5α cells. Chloramphenicol was used as instructed in the cultures, and all transformations were successful on the first try.
Both OD readings and Fluorescence readings were obtained for all 8 plasmids and the blank at 0, 2, 4, and 6 hours after transfer into media. There were two different colonies used for each plasmid, and four replicate measurements for each colony, resulting in a grand total of 576 measurements. Light was blocked by aluminum foil on the incubator glass to not disrupt the fluorescence.
The data obtained was then copied from the raw plate plate reading measurement sheet into the corrected sheet. Because no instrument correction was used, the data was corrected using the LUDOX and Fluorescein calibration factors in the corrected measure sheet. The arithmetic average data has been represented below graphically in Figures 3 and 4:
All data shows good replicability between colonies except for the first construct, which likely had some sort of error with the experimental technique after the transformation.