Browsing by Author "Landry, Brian P."

Now showing 1 - 4 of 4
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    An open-hardware platform for optogenetics and photobiology
    (Springer Nature, 2016) Gerhardt, Karl P.; Olson, Evan J.; Castillo-Hair, Sebastian M.; Hartsough, Lucas A.; Landry, Brian P.; Ekness, Felix; Yokoo, Rayka; Gomez, Eric J.; Ramakrishnan, Prabha; Suh, Junghae; Savage, David F.; Tabor, Jeffrey J.
    In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.
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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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    Phosphatase activity tunes two-component system sensor detection threshold
    (Springer Nature, 2018) Landry, Brian P.; Palanki, Rohan; Dyulgyarov, Nikola; Hartsough, Lucas A.; Tabor, Jeffrey J.
    Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.
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    Rewiring bacterial two-component systems by modular DNA-binding domain swapping
    (Springer Nature, 2019) Schmidl, Sebastian R.; Ekness, Felix; Sofjan, Katri; Daeffler, Kristina N-M; Brink, Kathryn R.; Landry, Brian P.; Gerhardt, Karl P.; Dyulgyarov, Nikola; Sheth, Ravi U.; Tabor, Jeffrey J.
    Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterizedﾠShewanella oneidensisﾠTCSs inﾠEscherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.