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. 2018 Jun 21;13(6):e0199432.
doi: 10.1371/journal.pone.0199432. eCollection 2018.

Quantification of bacterial fluorescence using independent calibrants

Jacob Beal  1 , Traci Haddock-Angelli  2 , Geoff Baldwin  3 , Markus Gershater  4 , Ari Dwijayanti  3 , Marko Storch  3 , Kim de Mora  2 , Meagan Lizarazo  2 , Randy Rettberg  2 ; with the iGEM Interlab Study Contributors
Affiliations

Quantification of bacterial fluorescence using independent calibrants

Jacob Beal et al. PLoS One. .

Abstract

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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Conflict of interest statement

The authors of this manuscript have read the journal’s policy and have the following competing interests: The authors received no specific commercial funding for this work. The following authors are employed by for-profit companies: Jacob Beal is employed by Raytheon BBN Technologies; Markus Gershater is employed by Synthace, and their work on this paper was thus indirectly supported by their salaries. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Constitutive fluorescence constructs measured in the 2016 iGEM Interlab Study, diagrammed using standard SBOL Visual symbols [9].
Fig 2
Fig 2. Precision effects of calibration for (a) bulk fluorescence and (b) flow cytometry, showing geometric standard deviation over the values reported from different laboratories (geometric mean over test devices ±1 geometric standard deviation).
Fig 3
Fig 3. Fold range of 95% confidence interval vs. data treatment for (a) bulk fluorescence and (b) flow cytometry.
Fig 4
Fig 4. Measured fluorescence of test devices for (a) bulk fluorescence, (b) flow cytometry, and (c) both types of instruments, normalized against the positive control.
In each box, red plus indicates mean, red line indicates median, top and bottom edges indicate 25th and 75th percentiles, and whiskers extend from 9%–91%.
See this image and copyright information in PMC

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