Difference between revisions of "Team:UCSC/InterLab"

Line 75: Line 75:
 
<br>
 
<br>
 
<mainp>
 
<mainp>
−
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.
+
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.
 
</mainp>
 
</mainp>
 +
 
<h2>Methods</h2>
 
<h2>Methods</h2>
−
<mainp>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. <mainp><br>
+
<mainp>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 <a target = "_blank" href="https://2017.igem.org/Competition/InterLab_Study">interlab study main page</a>.<mainp><br>
 +
 
 
<h3>Protocol</h3>  
 
<h3>Protocol</h3>  
−
<subp>The <a target="_blank" href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">iGEM Plate Reader Protocol</a> was followed for both absorbance and fluorescence calibrations, and for cell measurements.<subp>
+
 
 +
<subp>The <a target="_blank" href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">iGEM Plate Reader Protocol</a> was followed for calibrations of absorbance and fluorescence, and for cell measurements.</subp><br>
 +
 
 +
<subp>The <a target="_blank" href="http://parts.igem.org/Help:Protocols/Transformation#Single_Tube_Transformation_Protocol">iGEM Single Tube Transformation Protocol</a> was used for transformations.</subp>
 +
 
 
    
 
    
 
<h3>Calibrations</h3>
 
<h3>Calibrations</h3>
−
<subp>Absorbance at 600nm was measured for LUDOX S40 and H2O, to calibrate the conversion factor optical density (OD600) and absorbance at 600nm (Abs600). </subp><br>
+
<subp>Absorbance at 600nm was measured for LUDOX S40 and H2O and results were used in the provided iGEM spreadsheet to calibrate the conversion factor optical density (OD600) and absorbance at 600nm (Abs600). </subp><br>
 +
 
 +
<subp>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. </subp><br>  
  
−
<subp>A fluorescein standard curve was constructed plotting fluorescence vs uM fluorescein of 2:1 serial dilutions of 50uM fluorescein.</subp><br>
 
  
 
<h3>Cell Measurements</h3>
 
<h3>Cell Measurements</h3>
−
<subp>Measurements were performed according to the <a target="_blank" href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">iGEM Plate Reader Protocol</a>protocol. </subp>
+
<subp>Measurements were performed according to the <a target="_blank" href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">iGEM Plate Reader Protocol</a>. </subp>
−
<subp>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 LINK<a target=“_blank” href=“”>iGEM Interlab Study Plate Reader Protocol</a>.</subp><br>
+
 
 +
<subp>E. coli DH5α Inoue competent cells were transformed according to the iGEM single tube transformation protocol.
  
−
<subp>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? . . . </subp><br>
+
<subp>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.</subp><br>
  
−
<subp>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. </subp><br>
+
<subp>After dilutions, measurement t=0hours was taken, and the 16 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. </subp><br>
  
 
<h2>Flourescence Measurements:</h2>         
 
<h2>Flourescence Measurements:</h2>         

Revision as of 03:37, 4 October 2017

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 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 and results were used in the provided iGEM spreadsheet to calibrate the conversion factor optical density (OD600) and absorbance at 600nm (Abs600).
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, measurement t=0hours was taken, and the 16 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:

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.
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.

S P O N S O R S
S O C I A L
C O N T A C T
Jack Baskin School of Engineering
University of California, Santa Cruz
1156 High Street
Santa Cruz, California, 95064