Revision as of 04:11, 4 October 2017

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, 50uM stock solution of fluorescein was serially diluted by 2:1, and measured fluorescence vs uM 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 50uM and 25uM 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.

Figure A:

Figure B:

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, subp. e0150182, Mar. 2016.

@@ Line 341: / Line 341: @@
 <h2>Results</h2>
-<mainp>Figure standard curves used
+<mainp>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.</mainp>
+dilution assay of fluorescein stock solution in PBS buffer. The highest fluorescein concentrations 50uM and 25uM 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. </mainp>
 <center>
@@ Line 354: / Line 353: @@
 <caption>Figure B:</caption>
 </center>
+<br>
-<br>
 <h2>References:</h2>
 <mainp>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, subp. e0150182, Mar. 2016.<mainp>
@@ Line 362: / Line 362: @@
 <!--<html>
 <div class="column full_size judges-will-not-evaluate">
 <h3>★  ALERT! </h3>

Difference between revisions of "Team:UCSC/InterLab"