Browsing by Author "Sexton, John T."

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    FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units
    (American Chemical Society, 2016) Castillo-Hair, Sebastian M.; Sexton, John T.; Landry, Brian P.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical Biological Physics
    Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae Venus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond.
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    Multiplexing cell-cell communication
    (EMBO Press, 2020) Sexton, John T.; Tabor, Jeffrey J.
    The engineering of advanced multicellular behaviors, such as the programmed growth of biofilms or tissues, requires cells to communicate multiple aspects of physiological information. Unfortunately, few cell-cell communication systems have been developed for synthetic biology. Here, we engineer a genetically encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. Our design comprises multiplexer and demultiplexer sub-circuits constructed from a total of 12 CRISPRi-based transcriptional logic gates, an acyl homoserine lactone-based communication module, and three inducible promoters that enable small molecule control over the conversations. Experimentally parameterized mathematical models of the sub-components predict the steady state and dynamical performance of the full system. Multiplexed cell-cell communication has applications in synthetic development, metabolic engineering, and other areas requiring the coordination of multiple pathways among a community of cells.
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    Synthetic Biology Open Language Visual (SBOL Visual) Version 2.0
    (De Gruyter, 2018) Cox, Robert Sidney; Madsen, Curtis; McLaughlin, James; Nguyen, Tramy; Roehner, Nicholas; Bartley, Bryan; Bhatia, Swapnil; Bissell, Mike; Clancy, Kevin; Gorochowski, Thomas; Grünberg, Raik; Luna, Augustin; Le Novère, Nicolas; Pocock, Matthew; Sauro, Herbert; Sexton, John T.; Stan, Guy-Bart; Tabor, Jeffrey J.; Voigt, Christopher A.; Zundel, Zach; Myers, Chris; Beal, Jacob; Wipat, Anil
    People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.0 of SBOL Visual, which builds on the prior SBOL Visual 1.0 standard by expanding diagram syntax to include functional interactions and molecular species, making the relationship between diagrams and the SBOL data model explicit, supporting families of symbol variants, clarifying a number of requirements and best practices, and significantly expanding the collection of diagram glyphs.