Team:Lethbridge/InterLab
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.
The measurement was done in a plate reader. Because the 96 well format of the plate reader is convenient for multiple measurement, the methods are written from the perspective of 96 well format.
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.
Firstly, the standard curve for fluorescence using the sodium fluorescein reference material needs to be obtain. Since the reference material should have consistent result, the standard curve of fluorescein values can be used to calibrate the values in the plate reader. The instrument settings must be exactly the same as other labs to ensure the quality of the standard curve. Here is the list of settings that must be standardized; the amplitude of the signal collected; filters or monochromator settings; slit widths; gain settings; plates or cuvette type used; measurement from top or bottom (in plates); number of reads (integration time); orbital averaging. In order to further improve the design of the experiment, additional standard curve must be collected with different sensitivity settings. The additional standard curves will potentially improve the analysis of a cell based assays by matching the most suitable standard curve for the data.
For complete methods: https://2018.igem.org/Measurement/InterLab/Plate_Reader
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
Particle and Fluorescence Standard Curve protocol
The data collected with Molecular Devices Spectramax i3x in 96 well format. The linear relationship between absorbance and particle counts and the linear relationship between fluorescence and fluorescein are desired by the iGEM InterLab committee. However, the log scale graph of the particle standard curve (Figure 1 B) and the standard curve (Figure 2 A) has exponential and square root relationship respectively. The possible source of error would be consistent pipetting error and oversaturation of the detector.
Fluorescein standard curve
Fluorescein standard curve (log scale)
Standard curve of particles
Standard curve of particles (log scale)
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.
Average fluorescence per OD
Average fluorescence per particle