Interlab
Goal
We took part in the Fifth International Interlab Measurement Study which aims to determine if we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units instead of OD OD Optical density .
Materials
Plate reader: BioTek
Plate reader plates: clear plates
Devices:
Positive control: BBa_R0040
Negative control: BBa_I20270
Device 1: BBa_J364000
Device 2: BBa_J364001
Device 3: BBa_J364002
Device 4: BBa_J364007
Device 5: BBa_J364008
Device 6: BBa_J364009
Calibration material: LUDOX CL-X and Silica beads for absorbance and Fluorescein for fluorescence(provided in the iGEM distribution)
Microorganism: Escherichia coli DH5α strains
Protocol
In order to compare data from different labs, all the teams were asked to follow the protocol provided by iGEM HQ. These can be found at:
2018 Interlab Plate Reader ProtocolProtocols/Transformation
Results
Before we took the cell measurements, we made three sets of unit calibration measurements.
First, we used LUDOX CL-X as a single point reference to obtain a conversion factor to transform Abs600 data into a comparable OD600 measurement. The conversion factor turns to be 3.111.
Then, we used a dilution series of monodisperse silica microspheres provided in kit and measured the Abs600 of them to construct a standard curve of a particle concentration, which allows us to convert Abs600 to an estimated number of cells.
Fig 1. The particle standard curve obtained form the 2nd calibration experiment.
Last, we prepared a dilution series of fluorescein provided in kit and measure the fluorescence in our plate reader. By measuring these, we generated a standard curve of fluorescence for fluorescein concentration, which we used to convert the data we measured to equivalent fluorescein concen
Fig 2. The fluorescein standard curve form 3rd calibration experiment.
In cell measurements, we measured the fluorescence and Abs600 of all devices and blank samples at hour 0 and hour 6. The results are shown below:
Fig 3. Fluorescence raw values at different time points.
Fig 4. Abs600 raw values at different time points.
Fig 5. µM/OD600 at hour 6 for all devices.
Finally we calibrated OD600 to colony forming unit（CFU） counts by spading plate for a dilution series of all devices with a 0.1 OD600.
Table 1. Colony forming units per 0.1 OD600
| samples | dilution factor | CFU/mL | ||
|---|---|---|---|---|
| 8×104 | 8×105 | 8×106 | ||
| 1.1 | TNTC | 48 | 11 | 3.84E+07 |
| 1.2 | 248 | 41 | 10 | 3.28E+07 |
| 1.3 | 172 | 54 | 5 | 4.32E+07 |
| 2.1 | TNTC | 143 | 20 | 1.14E+08 |
| 2.2 | TNTC | 153 | 25 | 1.22E+08 |
| 2.3 | TNTC | 151 | 18 | 1.21E+08 |
| 3.1 | TNTC | 119 | 16 | 9.52E+07 |
| 3.2 | TNTC | 125 | 19 | 1.00E+08 |
| 3.3 | TNTC | 89 | 18 | 7.12E+07 |
| 4.1 | TNTC | 209 | 16 | 1.67E+08 |
| 4.2 | TNTC | 130 | 17 | 1.04E+08 |
| 4.3 | TNTC | 164 | 10 | 1.31E+08 |
Conclusion
According to our data, device 4 showed the best fluorescence results, much better than the positive control. Device 1 was the second one with the highest emission. Device 3 showed lowest fluorescence emission, even lower than the negative control. Compared to the strength of all 6 devices’ promoter provided on the http://parts.igem.org/Part:BBa_J23101, we found that device 5 showed a rather low emission which was not consistent with the efficiency of its promoter. Since there were some problems with the transformation of device 5 from the very beginning, so probably the low fluorescence emission has something to do with the plasmid sequence.
As for the conversion factor from OD to CFU, it is 9.51×108 CFU/mL in samples whose OD600 is 1 and Abs600 is 0.321 according to the conversion factor between OD600 and Abs600 while from the particle standard curve we obtained from the 2nd calibration experiment, the numbers of particles in samples whose Abs600 is 0.321 should be around 1.43×108. So there is still some difference between CFU and absorbance of cells in terms of computing the number of cells.
