INTERLAB STUDY

Overview

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 important 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, instrument specifications are listed below. Raw cell measurement data was imported into the excel sheets provided by the iGEM headquarters.

Protocol

The iGEM Plate Reader Protocol was followed for both absorbance and fluorescence calibrations, and for cell measurements.

Calibrations

Absorbance at 600nm was measured for LUDOX S40 and H2O, to calibrate the conversion factor optical density (OD600) and absorbance at 600nm (Abs600).

A fluorescein standard curve was constructed plotting fluorescence vs uM fluorescein of 2:1 serial dilutions of 50uM fluorescein.

Cell Measurements

Measurements were performed according to the iGEM Plate Reader Protocolprotocol.

E. coli DH5α Inoue competent cells were transformed according to the recommended iGEM Single Tube Transformation Protocol LINK. The serial dilutions of fluorescein were diluted according to the LINKiGEM Interlab Study Plate Reader Protocol.

Cells were grown overnight for 14 hours to a final optical density of ..... The cell cultures were then diluted according to the calculations in the dilution calculation sheet. Include sheet? . . .

At this point a measurement t=0 was taken, and the cell cultures were allowed to incubate for 6 hours, with 500mL aliquots taken every 2 hours for measurements. All aliquots were kept on ice, and transferred to the recommended format in the 96-well plates.

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:

Filters:
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

Figure standard curves used (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.

Other plots including graphical interpretations of results

References:

1. J. Beal, T. Haddock-Angelli, M. Gershater, K. d. Mora, M. Lizarazo, J. Hollenhorst, R. Rettberg, and i. I. S. Contributors, “Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli,” PLOS ONE, vol. 11, p. e0150182, Mar. 2016.

Team:UCSC/InterLab