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Revision as of 01:09, 24 September 2018

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 Protocol
Protocols/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

Disscussion

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