Interlab
Goal
We took part in the Fifth International Interlab Measurement Study that aims to clarify the possibility of reducing lab-to-lab variability in fluorescence measurements through 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 teams were asked to follow the protocol provided by iGEM HQ, and can be found at:
2018 Interlab Plate Reader ProtocolProtocols/Transformation
Results
Before performing cell measurements, we ruled out 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’ve constructed a dilution series of monodisperse silica microspheres provided in kit and measured the Abs600 of them. The results were used to construct a standard curve of a particle concentration that allows us to convert Abs600 to an estimated number of cells.
Fig 1. The particle standard curve obtained form the 2nd calibration experiment.
At last, we’ve prepared a dilution series of fluorescein provided in kit and measure the fluorescence in our plate reader. By analyzing the data, we generated a standard curve of fluorescence for fluorescein concentration, enabling us to convert the data we measured to equivalent fluorescein concentration.
Fig 2. The fluorescein standard curve form 3rd calibration experiment.
In cell measurements, we measured the fluorescence and Abs600 of all devices including 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 has shown the best fluorescence results which are even better than positive control while Device 1 was the second highest. On the contrary, Device 3 showed lowest fluorescence emission and even lower than negative control. Comparing with the strength of all 6 devices’ promoter provided on the 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 it is possible that the low fluorescence emission has something to do with the plasmid sequence.
As for the conversion factor from OD to CFU, we reach to the ratio of 9.51×108 CFU/mL in samples with OD600 = 1. And according to the conversion factor we calculated between OD600 and Abs600, when OD600 = 1, the corresponding Abs600=0.321. And based on the particle standard curve we obtained from the 2nd calibration experiment, the numbers of particles in samples with Abs600 = 0.321 should be around 1.43×108. So there is still difference between CFU and absorbance of cells in terms of computing the number of cells.
