Team:ETH Zurich/InterLab

InterLab Study

The Challenge

How close can the numbers be when fluorescence is measured all around the world?

Reliable and reproducible measurements are the fundamentals of science. However, up until this day, the comparison of fluorescence data remains a big challenge in synthetic biology. The InterLab study is a collaboration of iGEM teams around the world with the goal of determining the accuracy and robustness of GFP fluorescence data and comparing results. By following a uniform protocol and recording the resulting fluorescence in absolute units and its variability, the InterLab Study is aiming to improve the measurement tools available to the synthetic biology community.

This year we characterized eight plasmids:

After generating an OD600 reference point and a fluorescein standard curve, we transformed the test devices and measured the resulting fluorescence in a plate reader. Visit the iGEM InterLab Study page for more information.

Experiments

Experiment 1: OD600 Reference Point

OBJECTIVE
Use LUDOX-HS40 as a single point reference to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement.

MATERIALS

  • LUDOX-HS40 (Sigma Aldrich, Cat No 420816, Interlab Study Measurement Kit)
  • Distilled water (dH2O)
  • Greiner 96-well flat clear bottom black polystyrol plate (Greiner Bio One International GmbH, Germany)
  • Tecan Pro M1000 Plate Reader (Tecan Group Ltd., Switzerland)

METHODS

  • Add 100 uL of 100% LUDOX-HS40 in 4 wells
  • Add 100 uL of dH2O in 4 wells
  • Measure absorbance at 600 nm of all samples in standard measurement modes in instrument

MEASUREMENTS

  • Mode: absorbance
  • Wavelength: 600 nm
  • Number of flashes: 25
  • Settle time: 0 ms

RESULTS

Absorbance calibration
Figure 1. Using LUDOX-HS to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD600 measurement. R1-R4 = replicates 1 to 4, Mean = arithmetic mean
Experiment 2: Fluorescein Fluorescence Standard Curve

OBJECTIVE
Generate a standard curve of fluorescence for fluorescein concentration. Use the standard curve to correct the cell based readings to an equivalent fluorescein concentration. This concentration can then be converted into a concentration of GFP.

MATERIALS

  • 100 µM Fluorescein sodium salt (Sigma Aldrich, Cat No 46970, Interlab Study Measurement Kit)
  • 1x PBS (phosphate buffered saline)
  • Greiner 96-well flat bottom black polystyrol plate (Greiner Bio One International GmbH, Germany)
  • Tecan Pro M1000 Plate Reader (Tecan Group Ltd., Switzerland)

METHODS

  • Add 100 µL of 1x PBS in the wells A2:C12.
  • Add 200 µL of 100 µM fluorescein sodium salt solution into the wells A1, B1, C1.
  • Create serial dilutions of fluorescein by transferring 100 µL of solution starting from wells A1, B1, C1 to A2, B2, C2 until A11, B11, C11 wells are filled. Discard the extra 100 µL from A11, B11, C11 .
  • Measure fluorescence of all samples in standard measurement modes in instrument.

MEASUREMENTS

  • Mode: fluorescence bottom reading
  • Excitation wavelength: 488 nm ± 5 nm
  • Emission wavelength: 509 nm ± 5 nm
  • Gain: 60 (Manual)
  • Number of flashes: 50
  • Flash frequency: 400 Hz
  • Integration time: 20 µs
  • Lag time: 0 µs
  • Settle time: 0 ms
  • Shaking (orbital) duration: 10 s
  • Shaking (orbital) amplitude: 6 mm
  • Shaking (orbital) frequency: 120 rpm

RESULTS

Fluorescein calibration
Figure 2. Fluorescein Fluorescence Standard Curve shown in A) linear scale and B) log-log scale.
Experiment 3: Cell Measurement

OBJECTIVE
Determine GFP expression in E. coli K-12 DH5α cells transformed with different plasmids.

MATERIALS

    • Competent cells (E. coli strain DH5α)
    • LB (Luria Bertani) media (ThermoFisher Scientific, Switzerland)
    • LB Agar (Bacto-Agar, BD Biosciences, France) plates supplemented with 25 µg/mL Cloramphenicol (Sigma Aldrich, Germany)
    • 25 mg/mL Chloramphenicol dissolved in EtOH
    • 15 mL Greiner tubes (Greiner Bio One International GmbH, Germany)
    • 50 mL Falcon tubes covered in foil (Greiner Bio One International GmbH, Germany)
    • Incubator (ClimoShaker ISFX-1, Adolf Kühner AG, Switzerland)
    • Greiner 96-well flat bottom black polystyrol plate (Greiner Bio One International GmbH, Germany)
    • Tecan Pro M1000 Plate Reader (Tecan Group Ltd., Switzerland)

METHODS

  • Day 1: Transform chemically competent K-12 DH5α E. coli with the following plasmids:
    • 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
  • Day 2: Pick two colonies from each plate and inoculate it on 5-10 mL LB medium + chloramphenicol. Grow the cells overnight (16-18 hours) at 37 oC and 220 rpm.
  • Day 3: Measure OD600 of the overnight cultures. Dilute the cultures to a target OD600 of 0.02 in 12 mL LB medium + chloramphenicol in 50 mL falcon tube. Incubate at 37 oC and 220 rpm. Take 500 µL samples of the cultures at 0, 2, 4 and 6 hours of incubations. Measure OD and fluorescence.

MEASUREMENTS

  • As instructed, same instrument settings that we used in Experiments 1 and 2 were used.

RESULTS

Cell Measurement
Figure 3. Cell measurement. (1) = replicate 1, (2) = replicate 2, NC = negative control, PC = positive control, TD1-TD6 = test devices 1 to 6