Team:UCSC/InterLab
INTERLAB STUDY
Introduction
The InterLab Measurement Study is an international effort to increase reproducibility of biomedical research data [1]. Genetically engineered constructs are highly sensitive to environmental factors, and as well as the biological instruments being used. Since minimizing the discrepancies of environmental factors is difficult, the InterLab Study mitigates inconsistencies by focusing on the instruments used. The purpose of this year’s InterLab Study is to address variations in fluorescence measurements, particularly with respect to green fluorescent protein (GFP).
Every iGEM team is provided with the same constitutive constructs and protocols, and are required to transform into the same strain of E. coli (DH5α). Therefore, the only substantial variable is the machine used for fluorimetry measurements. Although teams are required to use a 96-well plate reader, there are many different makes and models of machines available for use. The InterLab Study generates data that allows the scientific community to better understand and identify sources of variation to increase the reproducibility of fluorescent measurements.
Methods
All measurements were taken using a using a Perkin Elmer EnVision 96-well format plate reader. Plates used were black with flat, clear bottoms. Plate reader specifications were saved with with EnVision Workstation version 1.09 and instrument specifications are listed below. Raw measurement data was imported into the excel sheets provided by the iGEM headquarters. The devices used can be found listed at the bottom of the interlab study main page.
Protocol
The iGEM Plate Reader Protocol was followed for calibrations of absorbance and fluorescence, and for cell measurements.
The iGEM Single Tube Transformation Protocol was used for transformations.
Calibrations
Absorbance at 600nm was measured for LUDOX S40 and H2O. The results were imported to the provided iGEM spreadsheet to calibrate the conversion factor between absorbance at 600nm (Abs600) and optical density (OD600).
To construct standard curves, 50µM stock solution of fluorescein was serially diluted by 2:1, and measured fluorescence vs µM fluorescein was plotted on both linear and logarithmic scales.
Cell Measurements
Measurements were performed according to the iGEM Plate Reader Protocol.
E. coli DH5α Inoue competent cells were transformed according to the iGEM single tube transformation protocol.
Cells were grown overnight for 14 hours, the optical densities were measured and imported into the excel dilution calculation sheet. Cell cultures were then diluted accordingly in 50mL falcon tubes covered in aluminum foil.
After dilutions, the 16 cell cultures were allowed to incubate for 6 hours, with 500mL aliquots taken at 0, 2, 4, and 6 hours for measurements. All aliquots were kept on ice, and transferred to the recommended format in the 96-well plates.
Detailed Instrument Settings
The fluorescence and absorbance settings are listed below.
Flourescence Measurements
| GFP IGEM | 4000065 |
|---|---|
| Top mirror | FITC |
| Bottom mirror | N/A |
| Exc. filter | FITC 485 |
| Using of excitation filter | Top |
| 2nd exc. filter | N/A |
| Using of 2nd excitation filter | Top |
| Ems. filter | FITC 535 |
| 2nd ems. filter | N/A |
| Measurement height | 6 mm |
| Number of flashes | 10 |
| Number of flashes integrated | 1 |
| PMT gain | 1 |
| Limits of excitation light | 1% |
| Range of excitation light | 100% |
| Reference AD gain | 2 |
| Reference signal | 388381 |
| Last edited | 9/21/17 11:34 |
| Last edited by | EnVision |
| Factory preset | No |
Absorbance 600 settings:
| Absorbance @ 600 | 2000005 |
|---|---|
| Exc. filter | Photometric 600 |
| Measurement height | 13 mm |
| Number of flashes | 10 |
| Number of flashes integrated | 1 |
| Limits of excitation light | 100% |
| Range of excitation light | 100% |
| Reference AD gain | 8 |
| Reference signal | 260565 |
| Continuous measurement | No |
| Last edited | 3/29/16 0:37 |
| Last edited by | EnVision |
| Factory preset | No |
Filters Used:
| FITC 485 | 102 |
|---|---|
| Filter type | Excitation |
| Description | X485 CWL=485nm BW=14nm Tmin=60% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved Fluorescence |
| Last edited | 10/21/10 13:14 |
| Last edited by | EnVision |
| Factory preset | Yes |
| FITC 535 | 206 |
| Filter type | Emission |
| Description | M535 CWL=535nm BW=25nm Tmin=50% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved FluorescenceLuminescence DELFIA - Time-resolved Fluorescence Fluorescence Intensity LANCE - Time-resolved Fluorescence |
| Last edited | 6/25/07 11:39 |
| Last edited by | Installation |
| Factory preset | Yes |
| Photometric 600 | 319 |
| Filter type | Excitation |
| Description | P600 CWL=600nm BW=8nm Tmin=40% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved FluorescenceLuminescence DELFIA - Time-resolved Fluorescence Fluorescence Intensity LANCE - Time-resolved FluorescenceAbsorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved Fluorescence |
| Last edited | 6/25/07 11:39 |
| Last edited by | Installation |
| Factory preset | Yes |
Results
Standard curves showing (a) linear and (b) logarithmic plots for the standard curve obtained from a 2:1 serial dilution assay of fluorescein stock solution in PBS buffer. The highest fluorescein concentrations 50µM and 25µM were not included in the curves, as they were outside the linear range of measurement. None of the cellular fluorescence measurements were off the scale of the standard curves.
Fluorescence measured per optical density was calculated by correcting for background, and dividing fluorescence readings by the optical density at each time point.
| Hour 0: | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Arith. Mean | Arith. Std.Dev. | Geo. Mean | Geo. Std. Dev. | |
|---|---|---|---|---|---|---|---|---|---|
| Negative Control (Colony 1) | 0.01 | 0.02 | -0.02 | 0.00 | 0.00 | 0.02 | #NUM! | #NUM! | |
| Negative Control (Colony 2) | -0.02 | -0.01 | -0.01 | 0.02 | 0.00 | 0.02 | #NUM! | #NUM! | |
| Positive Control (Colony 1) | 0.20 | 0.23 | 0.21 | 0.22 | 0.22 | 0.01 | 0.22 | 1.068 | |
| Positive Control (Colony 2) | 0.37 | 0.35 | 0.31 | 0.31 | 0.34 | 0.03 | 0.34 | 1.100 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 2.01 | 1.83 | 1.76 | 1.88 | 1.87 | 0.11 | 1.87 | 1.057 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 1.47 | 1.29 | 1.35 | 1.52 | 1.41 | 0.10 | 1.40 | 1.077 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.69 | 0.57 | 0.58 | 0.63 | 0.61 | 0.06 | 0.61 | 1.097 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.71 | 0.71 | 0.71 | 0.77 | 0.72 | 0.03 | 0.72 | 1.041 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.03 | 0.01 | 0.00 | 0.04 | 0.02 | 0.02 | #NUM! | #NUM! | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.01 | 0.02 | 0.02 | 0.00 | 0.01 | 0.01 | #NUM! | #NUM! | |
| Test Device 4: J23101+I13504 (Colony 1) | 0.30 | 0.33 | 0.28 | 0.27 | 0.30 | 0.03 | 0.30 | 1.104 | |
| Test Device 4: J23101+I13504 (Colony 2) | 0.24 | 0.25 | 0.21 | 0.28 | 0.25 | 0.03 | 0.24 | 1.133 | |
| Test Device 5: J23106+I13504 (Colony 1) | 0.05 | 0.05 | 0.09 | 0.11 | 0.07 | 0.03 | 0.07 | 1.521 | |
| Test Device 5: J23106+I13504 (Colony 2) | 0.06 | 0.06 | 0.11 | 0.07 | 0.07 | 0.03 | 0.07 | 1.366 | |
| Test Device 6: J23117+I13504 (Colony 1) | 0.02 | 0.00 | 0.01 | 0.02 | 0.02 | 0.01 | 0.01 | 2.261 | |
| Test Device 6: J23117+I13504 (Colony 2) | 0.00 | 0.00 | -0.01 | -0.01 | 0.00 | 0.01 | #NUM! | #NUM! | |
| Hour 2: | |||||||||
| Negative Control (Colony 1) | 0.01 | 0.00 | 0.00 | 0.01 | 0.01 | 0.00 | 0.01 | 2.252 | |
| Negative Control (Colony 2) | 0.01 | 0.00 | 0.01 | 0.00 | 0.00 | 0.01 | #NUM! | #NUM! | |
| Positive Control (Colony 1) | 0.41 | 0.39 | 0.37 | 0.35 | 0.38 | 0.03 | 0.38 | 1.071 | |
| Positive Control (Colony 2) | 0.52 | 0.53 | 0.48 | 0.52 | 0.51 | 0.02 | 0.51 | 1.046 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 2.13 | 2.19 | 1.87 | 1.90 | 2.02 | 0.16 | 2.02 | 1.081 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 1.48 | 1.50 | 1.58 | 1.64 | 1.55 | 0.07 | 1.55 | 1.049 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.57 | 0.55 | 0.53 | 0.52 | 0.54 | 0.02 | 0.54 | 1.041 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.59 | 0.59 | 0.60 | 0.61 | 0.60 | 0.01 | 0.60 | 1.014 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.01 | 0.01 | -0.01 | -0.01 | 0.00 | 0.01 | #NUM! | #NUM! | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.01 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 3.786 | |
| Test Device 4: J23101+I13504 (Colony 1) | 0.62 | 0.60 | 0.54 | 0.53 | 0.58 | 0.05 | 0.57 | 1.082 | |
| Test Device 4: J23101+I13504 (Colony 2) | 0.53 | 0.55 | 0.48 | 0.51 | 0.52 | 0.03 | 0.52 | 1.053 | |
| Test Device 5: J23106+I13504 (Colony 1) | 0.12 | 0.11 | 0.13 | 0.12 | 0.12 | 0.01 | 0.12 | 1.061 | |
| Test Device 5: J23106+I13504 (Colony 2) | 0.14 | 0.13 | 0.13 | 0.13 | 0.13 | 0.00 | 0.13 | 1.022 | |
| Test Device 6: J23117+I13504 (Colony 1) | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 2.111 | |
| Test Device 6: J23117+I13504 (Colony 2) | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | #NUM! | #NUM! | |
| Hour 4: | |||||||||
| Negative Control (Colony 1) | 0.00 | 0.00 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 3.138 | |
| Negative Control (Colony 2) | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 2.489 | |
| Positive Control (Colony 1) | 0.30 | 0.29 | 0.28 | 0.28 | 0.29 | 0.01 | 0.29 | 1.028 | |
| Positive Control (Colony 2) | 0.43 | 0.46 | 0.42 | 0.44 | 0.44 | 0.01 | 0.44 | 1.032 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 2.21 | 2.38 | 2.12 | 2.19 | 2.22 | 0.11 | 2.22 | 1.050 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 1.68 | 1.60 | 1.78 | 1.88 | 1.73 | 0.12 | 1.73 | 1.073 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.53 | 0.53 | 0.52 | 0.54 | 0.53 | 0.01 | 0.53 | 1.010 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.57 | 0.59 | 0.60 | 0.64 | 0.60 | 0.03 | 0.60 | 1.055 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.01 | 0.01 | 0.00 | 0.01 | 0.01 | 0.00 | 0.01 | 1.518 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.01 | 0.01 | 0.00 | 0.01 | 0.01 | 0.00 | 0.01 | 1.340 | |
| Test Device 4: J23101+I13504 (Colony 1) | 0.52 | 0.50 | 0.48 | 0.48 | 0.50 | 0.02 | 0.50 | 1.039 | |
| Test Device 4: J23101+I13504 (Colony 2) | 0.45 | 0.46 | 0.45 | 0.47 | 0.46 | 0.01 | 0.46 | 1.018 | |
| Test Device 5: J23106+I13504 (Colony 1) | 0.08 | 0.07 | 0.07 | 0.08 | 0.08 | 0.00 | 0.08 | 1.043 | |
| Test Device 5: J23106+I13504 (Colony 2) | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.00 | 0.08 | 1.031 | |
| Test Device 6: J23117+I13504 (Colony 1) | 0.01 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.952 | |
| Test Device 6: J23117+I13504 (Colony 2) | 0.00 | 0.00 | 0.00 | 0.01 | 0.00 | 0.00 | 0.00 | 1.604 | |
| Hour 6: | |||||||||
| Negative Control (Colony 1) | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | #NUM! | #NUM! | |
| Negative Control (Colony 2) | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.640 | |
| Positive Control (Colony 1) | 0.28 | 0.28 | 0.29 | 0.28 | 0.28 | 0.00 | 0.28 | 1.013 | |
| Positive Control (Colony 2) | 0.51 | 0.50 | 0.48 | 0.50 | 0.50 | 0.01 | 0.50 | 1.029 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 2.38 | 2.36 | 2.20 | 2.07 | 2.25 | 0.15 | 2.25 | 1.069 | |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 1.69 | 1.75 | 1.74 | 1.70 | 1.72 | 0.03 | 1.72 | 1.018 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.70 | 0.79 | 0.70 | 0.63 | 0.70 | 0.07 | 0.70 | 1.099 | |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.69 | 0.73 | 0.69 | 0.69 | 0.70 | 0.02 | 0.70 | 1.027 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.00 | 0.01 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 1.410 | |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.01 | 0.01 | 0.01 | 0.00 | 0.01 | 0.00 | 0.01 | 1.267 | |
| Test Device 4: J23101+I13504 (Colony 1) | 0.47 | 0.51 | 0.53 | 0.51 | 0.50 | 0.02 | 0.50 | 1.047 | |
| Test Device 4: J23101+I13504 (Colony 2) | 0.41 | 0.42 | 0.41 | 0.41 | 0.41 | 0.01 | 0.41 | 1.015 | |
| Test Device 5: J23106+I13504 (Colony 1) | 0.07 | 0.06 | 0.07 | 0.07 | 0.07 | 0.00 | 0.07 | 1.065 | |
| Test Device 5: J23106+I13504 (Colony 2) | 0.10 | 0.10 | 0.09 | 0.10 | 0.10 | 0.00 | 0.10 | 1.023 | |
| Test Device 6: J23117+I13504 (Colony 1) | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.306 | |
| Test Device 6: J23117+I13504 (Colony 2) | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.630 |
