Difference between revisions of "Team:UCSC/InterLab"
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| − | E. coli DH5α Inoue competent cells were transformed according to the recommended iGEM Single Tube transformation protocol: <a href="http://parts.igem.org/Help:Protocols/Transformation#Single_Tube_Transformation_Protocol" Single Tube Protocol | + | E. coli DH5α Inoue competent cells were transformed according to the recommended iGEM Single Tube transformation protocol: <a href="http://parts.igem.org/Help:Protocols/Transformation#Single_Tube_Transformation_Protocol">Single Tube Protocol</a>. Measurements were taken using a Perkin Elmer Envision 96-well format plate reader. Data was imported into the excel data sheets provided by the iGEM headquarters. The serial dilutions of fluorescein were diluted according to protocol, and the measurements used to construct standard curves. The 50uM and 25uM fluorescein concentrations were not included in the standard curve, as they were outside the linear range of the detector. No device had GFP expression causing readings outside the range of the standard curve. |
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Revision as of 03:30, 1 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 important data that allows the scientific community to better understand and identify sources of variation to increase the reproducibility of fluorescent measurements.
Methods
E. coli DH5α Inoue competent cells were transformed according to the recommended iGEM Single Tube transformation protocol: Single Tube Protocol. Measurements were taken using a Perkin Elmer Envision 96-well format plate reader. Data was imported into the excel data sheets provided by the iGEM headquarters. The serial dilutions of fluorescein were diluted according to protocol, and the measurements used to construct standard curves. The 50uM and 25uM fluorescein concentrations were not included in the standard curve, as they were outside the linear range of the detector. No device had GFP expression causing readings outside the range of the standard curve.
The optical density was measured at each time point, to provide a useful comparison of fluorescence vs optical density. This translates to the amount of GFP produced by a given number of cells.
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 bufferOther 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.
