In order to advance reproducibility of fluorescence measurements, the iGEM competition decided to engage the network of several hundred laboratories across the globe in one of the largest inter-laboratory studies ever conducted: the InterLab Study. For now the fourth consecutive year, the InterLab Study aims to improve comparability of GFP fluorescence measurements by developing a standard measurement protocol – and this year, the diagnost-x Team took part in this effort. This year’s specific aim was to establish a protocol to compare GFP measurements on different models of plate readers. Furthermore, the GFP-expressing constructs sent out by the iGEM committee contained different prokaryotic ribosome-binding site devices (more specifically, bicistronic designs known as BCDs) in order to enhance precision and reliability of gene expression.
Methods & Protocols
1. Setup Parameters (Plate Reader)
Plate Reader: Tecan Infinite 200 Pro
Abs OD 600
- Wavelengths: 600 nm
- Bandwidth: 10 nm
- Resting time: 0 s
- Number of flashes: 25
- Optics: Top
Fluorescence Excitation 485nm
- Excitation wavelength: 485 nm
- Excitation bandwidth: 20 nm
- Emission wavelength: 535 nm
- Emission bandwidth: 25 nm
- Mirror: Dichroic 510 nm
- Gain: 30
- Number of flashes: 35
- Integration time: 40 µs
- Resting time: 0 s
- Optics: Top /li>
2. OD 600 Reference Point
- 1 ml LUDOX
- 96 well plate
- 1. Add 100 μl LUDOX into wells A1, B1, C1, D1
- 2. Add 100 μl of H2O into wells A2, B2, C2, D2
- 3. Measure absorbance 600 nm of all samples as indicated in Setup Parameters
- 4. Use measurements to calculate correction factor
3. Fluorescence standard curve protocol
- 1 Fluorescein
- 10 ml 1xPBS
- 96 well plate
- 1. Spin down fluorescein stock tube
- 2. Prepare 2x fluorescein stock solution (100 μM) resuspending fluorescein 1 ml 1xPBS
- 3. Dilute 500 µl of the 2x fluorescein stock solution with 500 µl 1xPBS to make a 1x fluorescein solution (50 µM)
- 4. Make a serial dilution of fluorescein by taking the following steps:
- a. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
- b. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
- c. Transfer 100 μl of fluorescein stock solution from A1 into A2
- d. Mix A2 (by pipetting up and down), transfer 100 μl into A3
- e. Mix A3, transfer 100 μl into A4
- f. Mix A4, transfer 100 μl into A5
- g. Mix A5, transfer 100 μl into A6
- h. Mix A6, transfer 100 μl into A7
- i. Mix A7, transfer 100 μl into A8
- j. Mix A8, transfer 100 μl into A9
- k. Mix A9, transfer 100 μl into A10
- l. Mix A10, transfer 100 μl into A11
- m. Mix A11, transfer 100 μl into liquid waste
- 5. Repeat dilution series for rows B, C, D
- 6. Measure fluorescence of all samples as indicated above
- 7. Use data to calculate fluorescence standard curve
Cell measurement protocol
- Competent cells (E. coli strain DH5α)
- LB medium
- Chloramphenicol (stock concentration 25 mg/ml dissolved in EtOH - working stock 25 µg/ml)
- 50 ml Falcon tubes
- Incubator at 37°C
- 1.5 ml eppendorf tubes
- 96 well plate
- Devices from InterLab Measurement Kit:
- Positive control
- Negative control
- Test Device 1: J23101+I13504
- Test Device 2: J23106+I13504
- Test Device 3: J23117+I13504
- Test Device 4: J23101.BCD2.E0040.B0015
- Test Device 5: J23106.BCD2.E0040.B0015
- Test Device 6: J23117.BCD2.E0040.B0015
- Each of the Plasmids from the InterLab Measurement Kit was resuspended in 10 µl of H2O (distilled water).
- The plasmids were then transformed into competent DH5α-cells, using the the 96-well format transformation protocol recommended by iGEM Headquarters, and cells were plated on LB medium + Chloramphenicol plates and incubated overnight at 37°C.
- Two colonies were picked from each transformation and inoculated in 8 ml of LB medium + Chloramphenicol, resulting in a final 16 sample cultures.
- These cultures were grown overnight at 37°C and 220 rpm.
- Measure OD600 of the overnight cultures.
- Cultures were diluted to a target OD of 0.02 according to the dilution sheet provided by iGEM HQ.
- Cultures were re-incubated at 37°C and 220 rpm.
- 500 μL samples of the cultures were taken at 0, 2, 4, and 6 hours of incubation and placed on ice.
- After sample collection, samples were placed in a 96-well plate and both OD 600 and fluorescence were measured using the setup indicated below.
Fluorescence Standard Curve
Cell measurement: Absorption at 600nm
Cell measurement: Flourescence
Ex 485 nm/Em 535 nm
In order to really interpret the results of this study and judge its outcome, of course the results of all participating labs are needed. However, for our contribution to the 2017 InterLab study will subsequently try to sum up the results obtained by our team. In terms of standardization and calibration, we established a correction factor for Abs 600 measurements via an OD-600 Reference Point (LUDOX-HS40). Furthermore, we set up several standard curves for fluorescence measurements with GFP (Excitation 485 nm, Emission 535 nm). These different standard curves varied in the applied gain factor as well as in the integration time window. For our cell measurements, we then used a set of parameters that ensured all measured values to be in the range of the standard curve. The curve shown above represents this best set of parameters, but still is not ideal: we did not find a set of standard parameters that both ensured no value to be outside standard-curve-range and at the same time showed a perfect linear correlation on log scale. In the cell measurements protocol, our results show that some of the devices indeed produce a fluorescent product within the 18 to 24 hours after picking a transformed colony. However, only five devices (shown in green in the fluorescence graph) exhibited higher levels of fluorescence. This is partly due to a lack of viable cells: as can be seen from the Abs-600 graph, several colonies, highlighted in red, did not continue to grow after the dilution. But also some colonies with high absorbance, and thus a presumed high growth, did not exhibit fluorescence. In our experiment, only the positive control and Test Device 1, 2, and 4 lead to expression of a fluorescent product. We cannot estimate on the expression levels in Test Device 5 and 6, as growth of the cultures was low.