Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs

The iGEM Interlab Study Contributors, 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

*Corresponding author for this work

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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.
Original languageEnglish
Article numbere0252263
Number of pages15
JournalPLoS ONE
Issue number6
Publication statusPublished - 7-Jun-2021
Externally publishedYes

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