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Quantification of bacterial fluorescence using independent calibrants

Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific...

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Autores principales: Beal, Jacob, Haddock-Angelli, Traci, Baldwin, Geoff, Gershater, Markus, Dwijayanti, Ari, Storch, Marko, de Mora, Kim, Lizarazo, Meagan, Rettberg, Randy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013168/
https://www.ncbi.nlm.nih.gov/pubmed/29928012
http://dx.doi.org/10.1371/journal.pone.0199432
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author Beal, Jacob
Haddock-Angelli, Traci
Baldwin, Geoff
Gershater, Markus
Dwijayanti, Ari
Storch, Marko
de Mora, Kim
Lizarazo, Meagan
Rettberg, Randy
author_facet Beal, Jacob
Haddock-Angelli, Traci
Baldwin, Geoff
Gershater, Markus
Dwijayanti, Ari
Storch, Marko
de Mora, Kim
Lizarazo, Meagan
Rettberg, Randy
author_sort Beal, Jacob
collection PubMed
description Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.
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spelling pubmed-60131682018-07-06 Quantification of bacterial fluorescence using independent calibrants Beal, Jacob Haddock-Angelli, Traci Baldwin, Geoff Gershater, Markus Dwijayanti, Ari Storch, Marko de Mora, Kim Lizarazo, Meagan Rettberg, Randy PLoS One Research Article Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units. Public Library of Science 2018-06-21 /pmc/articles/PMC6013168/ /pubmed/29928012 http://dx.doi.org/10.1371/journal.pone.0199432 Text en © 2018 Beal et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Beal, Jacob
Haddock-Angelli, Traci
Baldwin, Geoff
Gershater, Markus
Dwijayanti, Ari
Storch, Marko
de Mora, Kim
Lizarazo, Meagan
Rettberg, Randy
Quantification of bacterial fluorescence using independent calibrants
title Quantification of bacterial fluorescence using independent calibrants
title_full Quantification of bacterial fluorescence using independent calibrants
title_fullStr Quantification of bacterial fluorescence using independent calibrants
title_full_unstemmed Quantification of bacterial fluorescence using independent calibrants
title_short Quantification of bacterial fluorescence using independent calibrants
title_sort quantification of bacterial fluorescence using independent calibrants
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013168/
https://www.ncbi.nlm.nih.gov/pubmed/29928012
http://dx.doi.org/10.1371/journal.pone.0199432
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