Toward Quantitative Comparison of Fluorescent Protein Expression Levels via Fluorescent Beads

Establishing an e↵ective engineering discipline always requires standardized and comparable units of measurement. Such measurements serve as a means of communication between the people and machines interacting with a project, ensure compatibility between components, and allow prediction of the results of design decisions. Regulating gene expression is foundational for organism engineering, and flow cytometry is an excellent means of quantifying large numbers of single cell gene expression measurements. At present, however, flow cytometry data is still often acquired in arbitrary or relative units, without standardizing the measurement by comparison to an independent reference material (i.e., one enabling precise calibration of measurements). Some have proposed standardizing to a biological cultured reference material (e.g., [3]), but fluorescence from such materials varies strongly, unpredictably, and often not proportional to the samples it is intended to be a reference for, thus resulting in a large degree of uncertainty in measurement. In contrast, stable reference materials have been developed, in the form of beads with a defined fluorescence quantified in terms of molecules of equivalent reference fluorophores (ERF; alternately MEF or ME[fluorophore]) [5]. These reference materials have been primarily employed in medical applications of flow cytometry, which typically use a small number of standard dyes rather than a wide range of fluorescent protein variants, and where the goals of measurement are typically focused on the “digital” goal of classifying cells into distinct populations, rather than the more“analog”goal of precisely quantifying levels of gene expression. Fluorescent beads have already been used as a reference material for engineering gene expression in a number of studies, including making high-precision circuit predictions (e.g., [2]), engineering novel biological sensors (e.g., [4]), and debugging circuit design problems (e.g., [1]). We now aim to validate these methods through interlaboratory studies and to develop supporting methods and recommended practices that will simplify widespread adoption of well-defined units in flow cytometry, thus accelerating scientific development and simplifying the engineering of biological organisms.