Team:SDU-CHINA/InterLab
Interlab
The InterLab protocol aims to address key issues in synthetic biology by providing researchers with detailed protocol and data analysis. In our case, we measure the absolute units for measurements of GFP in a plate reader. We were invited to participate in the Fifth International Interlaboratory Measurement Study. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. In order to compute the cell count in our samples, we use two orthogonal approaches:
- Converting between absorbance of cells and absorbance of a known concentration of beads.
- Counting colony-forming units (CFUs) from the sample.
By using these two approaches, we will be able to determine how much they agree with each other, and whether using one (or both) can help to reduce lab-to-lab variability in measurements.
We followed the protocol provided by the committee and finished all the measurement during July.
Day 1: We successfully transformed Escherichia coli DH5α with following plasmids:
| Device | Part 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_364001 | Kit Plate 7 | Well 2H |
| Test Device 3 | BBa_364002 | Kit Plate 7 | Well 2J |
| Test Device 4 | BBa_364007 | Kit Plate 7 | Well 2L |
| Test Device 5 | BBa_364008 | Kit Plate 7 | Well 2N |
| Test Device 6 | BBa_364008 | Kit Plate 7 | Well 2P |
Day 2: We picked 2 colonies respectively from every transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. Then the cells were cultured overnight (16-18 hours) at 37°C and 220 rpm.
Day 3: Firstly, we took three calibration measurements: an OD 600 reference point, a particle standard curve, and a fluorescein standard curve.
Secondly, we did cell measurement. From 0 hour to 6 hour, we did CFU measurement and coated all 36 plates. After that, we measured Abs600 and fluorescence of the cultures in the 96 wells plate in the plate reader.
Day 4: We counted the colonies on every plate with fewer than 300 colonies. Our experimental data can be seen here.
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