Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results.

The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).

Overview

Two approaches were used:

1. Converting between absorbance of cells to absorbance of a known concentration of beads.
Absorbance is a measure of how light is scattered by cells in liquid, which can be used to estimate the concentration of cells in the sample. Assuming that silica beads approximately the same size and shape of E. coli cells would scatter the light in a similar way, we can use the absorbance measurement of a known concentration of beads to produce a standardized “equivalent concentration of beads” measurement.

2. Counting colony-forming units (CFUs) from the sample.
Simply, growing cells on a plate and counting the number of colonies on the plate can determine the amount of cells in a sample of liquid. The concentration of the sample can be obtained with the number of singly grown cells in the plate, which indicates the amount of cells, and the volume of media that is plated out. The positive and negative control samples will be used to calculate a conversion factor from absorbance to CFU.

For complete methods, refer to: https://2018.igem.org/Measurement/InterLab/Plate_Reader

Our Equipment

A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.

Calibration

1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a conversion factor to transform absorbance data into a comparable OD measurement.

2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. The size and optical characteristics of these microspheres are similar to cells and there is a known amount of particles per volume. This standard curve was later used to convert absorbance measurements to an estimated number of cells.

3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert Cell-based readings to an equivalent fluorescein concentration.

Cell Measurement Protocol

Briefly, we measured the raw absorbance and fluorescence of diluted cells (transformed with the below testing devices) at 0 hours and after 6 hours of incubation at 37°C.

Results

We expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.

The conversion of average fluorescence to reference unit was designed to reduce the impact of unorganized unit system.
For fluorescence to OD conversion, fluorescence was converted to absorbance and to OD.
For fluorescence to particle conversion, Molecules of Equivalent of Fluorescence (MEFL) was converted to absorbance and to particles.
The trend showed that except device 3, all other devices contained fluorescence part and the device 2 and 5 had increased fluorescence part after 6 hours, but device 1,4, and 6 had decreased fluorescence part after 6 hours.