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.
Highlights
IntroductionS., Haddock-Angelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Telmer, C., Vignoni, A., Tennant, R., and Rutten, P
As there is no common format for microplate reader data, a function to parse the data into a standard format is required
Size calibration[26] currently available in other flow cytometry packages[5] can be added, which may be of particular use in order to understand changes or differences in morphology of different bacterial strains
Summary
S., Haddock-Angelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Telmer, C., Vignoni, A., Tennant, R., and Rutten, P. Calibration Protocol - Plate Reader Fluorescence Calibration V.3 https://www.protocols.io/view/calibration-protocolplate-reader-fluorescence-cal-6zrhf[56] (accessed Jul 8, 2020). S., Farny, N., Tennant, R., and Rutten, P. Calibration Protocol - Plate Reader Abs[600] (OD) Calibration with Microsphere Particles V.2 https://www.protocols. H. (2016) Principles for Designing Synthetic Microbial Communities. P. (2020) Single strain control of microbial consortia. M., HaddockAngelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Vignoni, A., and Tennant, R. IGEM Calibration Protocol - Red Fluorescent Proteins in Plate Readers https://www.protocols.io/view/igemcalibration-protocol-red-fluorescent-proteins-bcdjis4n. (2020) FlowStats: Statistical Methods for the Analysis of Flow Cytometry Data, Bioconductor.
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