INTERLAB STUDY

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. In 2018, GFP measurement is developed by the Measurement Committee to be experimented. GFP is the most common measurement marker in synthetic biology which is widely available in synthetic biology laboratories. Previously, Fluorescence, from GFP, data was collected in arbitrary settings which caused the technical difficulties in data comparison and interpretation. The purpose of the 2018 InterLab study is to standardize fluorescence measurement with a detailed protocol and data analysis form that produces absolute units for measurement in a plate reader.

Question

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

Methods

Overview

To answer this question, two approaches were used:

1. Converting between absorbance of cells to absorbance of a known concentration of beads
Absorbance measurement uses the light scattering by a sample of cells in liquid.The variable in this measurement is the concentration of the cell. With assumption that the silica beads at the size of typical E.coli cells would scatter the light in similar way, the absorbance measurement with known concentration of the beads can be standardized as the “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. 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.

2. 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

3. 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.

Cell Measurement Protocol

1. 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.

2. 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

3. 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.

Testing Devices

The negative control did not contain fluorescent DNA parts and positive control is the fluorescent beads with known value. From the fluorescence values of two controls, the fluorescence value of each devices were calculated. Additionally, the kit plate and well locations were also pre-determined to minimize the detecting errors between trials.

Device	Pert number	Plate	Location
Negative Control	BBa_R0040	Kit Plate 7	Well 2D
Positive Control	BBa_I20270	Kit Plate 7	Well 2B
Test Device 1	BBa_J364000	Kit Plate 7	Well 2F
Test Device 2	BBa_J364001	Kit Plate 7	Well 2H
Test Device 3	BBa_J364002	Kit Plate 7	Well 2J
Test Device 4	BBa_J364007	Kit Plate 7	Well 2L
Test Device 5	BBa_J364008	Kit Plate 7	Well 2N
Test Device 6	BBa_J364009	Kit Plate 7	Well 2P

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.