Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

METHODS OVERVIEW:

The measurements were done in a plate reader. Because the 96 well format of the plate reader is convenient for multiple measurements, the methods are written from the perspective of 96 well format. The 96 well plates were clear bottomed plates. All plate reading was done using a Molecular Devices Spectramax i3x. This device has variable temperature settings, pathlength correction and can measure both absorbance and fluorescence. All GFP measurements were taken at wavelengths of 532/25 for emission and 485/20 for excitation. We did not perform the optional flow cytometry experiment as our flow cytometer is difficult to use, requiring the efforts of a trained technician, making it impractical for its use in this study.

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CALIBRATION:

• Measured a standard curve for fluorescein to allow this data to be standardized with the data from other iGEM labs. Fluorescein displays similar excitation and emission as GFP and could also be read by the plate reader. This allowed for measurements of GFP fluorescing cells to to be transformed into similar fluorescein readings.
• Followed a LUDOX protocol to serve as a reference point that functioned as a conversion factor. This conversion factor allowed for the absorbance values taken at 600 nm to be converted into OD600 measurements.
• A plate was prepared with silica microspheres and read by the plate reader at 600 nm. These microspheres have similar functional characteristics to cells that allows for the absorbance measurements to accurately estimate cell counts. Our measurements for this step can be considered flawed as we neglected to perform the initial dilution of the samples before preparing the plate. As well, this sample was not recorded at the correct wavelength. Unfortunately, due to the nature of the error, and the limited quantity of the reagent, we depleted our supply and could not redo the experiment.




CELL MEASUREMENT PROTOCOL:

Day 1

• Transformation of Colonies
The making of competent cells and transformation protocol were obtained from the iGEM protocol archive. Plasmids were resuspended in 10uL of ddH2O and transformed into DH5-alpha E.Coli cells. Each plate was incubated overnight.

Day 2

• Culture Growth
A pair of colonies were chosen from each plate and each was placed into a test tube containing 5mL of LB medium and 5uL 1000x Chloramphenicol. These test tube colonies were then incubated overnight on a shaker at 37 degrees.



Day 3

• Fluorescence Assay
A 1:10 dilution was made of each colony and the absorbance was measured at 600 nm using the faculty plate reader. These readings were used to determine the dilution required for the colonies to have a concentration of 0.02. A diluted cultured was made for each sample and placed on ice. Further culture samples were made and placed into a 37 degree incubator for six hours. Once this was complete the samples were placed into individual wells on a 96 well plate. The fluorescence and absorbance was measured for each well. We found that the interlab study was a good way for us to learn the lab techniques that we used during the development of our own project. However, it seemed challenging to manipulate the bacteria by dilutions as specified in the methods, as our results seemed to vary.



RESULT