FlopR: An Open Source Software Package for Calibration and Normalization of Plate Reader and Flow Cytometry Data

Abstract

The measurement of gene expression using fluorescence markers has been a cornerstone of synthetic biology for the past two decades. However, the use of arbitrary units has limited the usefulness of these data for many quantitative purposes. Calibration of fluorescence measurements from flow cytometry and plate reader spectrophotometry has been implemented previously, but the tools are disjointed. Here we pull together, and in some cases improve, extant methods into a single software tool, written as a package in the R statistical framework. The workflow is validated using Escherichia coli engineered to express green fluorescent protein (GFP) from a set of commonly used constitutive promoters. We then demonstrate the package’s power by identifying the time evolution of distinct subpopulations of bacteria from bulk plate reader data, a task previously reliant on laborious flow cytometry or colony counting experiments. Along with standardized parts and experimental methods, the development and dissemination of usable tools for quantitative measurement and data analysis will benefit the synthetic biology community by improving interoperability.

Single strain control of microbial consortia

“…1 a). The engineered strain is further weakened through the constitutive expression of mCherry from one of the plasmids (p63_AF043), which also allows the separation of the engineered and competitor strains in the plate reader 40 . The competitor strain excludes the engineered strain when the bacteriocin is mutationally inactivated 41 , but is outcompeted with complete bacteriocin expression (Fig.…”

“… a The two strains are co-cultured in a microtitre plate and the subpopulation fractions are determined using fluorescent proteins in the engineered strain and the FlopR software 40 , allowing us to track the population dynamics over time. b The blue shaded area shows the period during which the competitor ratio is above its initial ratio; the orange shaded area shows the same for the engineered strain.…”

“…Measurements of absorbance (600 nm and 700 nm) and fluorescence (CFP: ex = 430 nm, em = 490 nm, GFP: ex = 485 nm, em = 530 nm, mCherry: ex = 575 nm, em = 620 nm) were taken every 20 min using Tecan Spark Control software. Data were processed using FlopR 40 to produce population and subpopulation curves, and plotted using R 60 with ggplot2 61 and dplyr 62 .…”

“…The fluorescence channels are detailed in Supplementary Table 6 . The flow cytometry data was processed using FlopR 40 to remove debris and doublets, and Hill functions were fitted using RStan 65 .…”

Measurement Techniques to Resolve and Control Population Dynamics of Mixed-Culture Processes

“…First, the raw signals have to be converted into units, which can be universally compared across any device. This can be done by referencing arbitrary unit measurements from different instruments and scales to the signal intensity derived from defined standards, such as fluorescein [113]. Finally, these defined units can be correlated to a mass, such as the cell dry weight of biomass.…”