Beal, Jacob and Baldwin, Geoff S. and Farny, Natalie G. and Gershater, Markus and Haddock-Angelli, Traci and Buckley-Taylor, Russell and Dwijayanti, Ari and Kiga, Daisuke and Lizarazo, Meagan and Marken, John and de Mora, Kim and Rettberg, Randy and Sanchania, Vishal and Selvarajah, Vinoo and Sison, Abigail and Storch, Marko and Workman, Christopher T. (2021) Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs. PLoS ONE, 16 (6). Art. No. e0252263. ISSN 1932-6203. doi:10.1371/journal.pone.0252263. https://resolver.caltech.edu/CaltechAUTHORS:20210607-115055955
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Abstract
Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.
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| Additional Information: | This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Received: February 8, 2021; Accepted: May 11, 2021; Published: June 7, 2021. This document does not contain technology or technical data controlled under either the U.S. International Traffic in Arms Regulations or the U.S. Export Administration Regulations. Partial support for this work was provided by NSF Expeditions in Computing Program 469 Award #1522074 as part of the Living Computing Project. Funder URL: https://www.nsf.gov/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The following authors are employed by for-profit companies: Jacob Beal is employed by Raytheon BBN Technologies; Markus Gershater and Vishal Sanchania are employed by Synthace. These companies provided support in the form of salaries for these authors, 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.” Data Availability: All relevant data are within the manuscript and its Supporting information files. Competing interests: 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 and Vishal Sanchania are 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. Author Contributions: Conceptualization: Jacob Beal, Geoff S. Baldwin, Natalie G. Farny, Markus Gershater, Traci Haddock-Angelli, Meagan Lizarazo, Kim de Mora, Randy Rettberg. Data curation: Jacob Beal, Natalie G. Farny, Traci Haddock-Angelli, Vinoo Selvarajah. Formal analysis: Jacob Beal. Investigation: Geoff S. Baldwin, Natalie G. Farny, Markus Gershater, Traci Haddock-Angelli, Russell Buckley-Taylor, Ari Dwijayanti, Daisuke Kiga, John Marken, Vishal Sanchania, Vinoo Selvarajah, Abigail Sison, Marko Storch, Christopher T. Workman. Methodology: Jacob Beal, Geoff S. Baldwin, Natalie G. Farny, Markus Gershater, Traci Haddock-Angelli, Russell Buckley-Taylor, Ari Dwijayanti, Daisuke Kiga, John Marken, Vishal Sanchania, Vinoo Selvarajah, Abigail Sison, Marko Storch, Christopher T. Workman. Project administration: Jacob Beal, Natalie G. Farny, Traci Haddock-Angelli. Writing – original draft: Jacob Beal, Natalie G. Farny. Writing – review & editing: Jacob Beal, Geoff S. Baldwin, Natalie G. Farny, Markus Gershater, Traci Haddock-Angelli, Russell Buckley-Taylor, Ari Dwijayanti, Daisuke Kiga, Meagan Lizarazo, John Marken, Kim de Mora, Randy Rettberg, Vishal Sanchania, Vinoo Selvarajah, Abigail Sison, Marko Storch, Christopher T. Workman. | ||||||||
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| Issue or Number: | 6 | ||||||||
| DOI: | 10.1371/journal.pone.0252263 | ||||||||
| Record Number: | CaltechAUTHORS:20210607-115055955 | ||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20210607-115055955 | ||||||||
| Official Citation: | Beal J, Baldwin GS, Farny NG, Gershater M, Haddock-Angelli T, Buckley-Taylor R, et al. (2021) Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs. PLoS ONE 16(6): e0252263. https://doi.org/10.1371/journal.pone.0252263 | ||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||
| ID Code: | 109422 | ||||||||
| Collection: | CaltechAUTHORS | ||||||||
| Deposited By: | George Porter | ||||||||
| Deposited On: | 08 Jun 2021 14:17 | ||||||||
| Last Modified: | 16 Nov 2021 19:36 |
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