Quantification of bacterial fluorescence using independent calibrants

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

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calibration
  • Confidence Intervals
  • Escherichia coli / metabolism*
  • Flow Cytometry
  • Fluorescence

Grants and funding

Partial support for this work was provided by NSF Expeditions in Computing Program Award #1522074 as part of the Living Computing Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Raytheon BBN Technologies and Synthace provided support in the form of salaries for authors JB and MG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section.