Browsing by Author "Ekness, Felix"

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    A Synthetic Biology Approach to Engineering Bacterial Two- Component Systems for Sensor Development and Discovery of Anti-Virulence Agents
    (2019-12-06) Ekness, Felix; Tabor, Jeffrey J; Matthews, Kathleen
    Bacterial two-component systems (TCSs) are the largest family of signal transduction pathways that enable bacteria to sense a diversity of stimuli including small peptides, environmental pollutants, and light. Canonical TCSs are composed of a transmembrane sensor histidine kinase (SK) that converts stimulus detection into phosphorylation of a cognate response regulator (RR). Upon phosphorylation, the cytoplasmic RR binds target output promoters, hereby modulating gene expression. TCSs are valuable sensors for synthetic biology due to their diverse sensing capabilities and straightforward transduction of detected stimulus into transcriptional regulation. TCSs are also emerging targets for novel therapeutic development due to their extensive role in regulating bacterial virulence and antibiotic resistance. Although TCSs are exciting sensors for synthetic biology and targets for therapeutic applications, most TCSs remain difficult to harness for applications and study due to output promoters that are unknown, subject to cross- regulation, or silent in heterologous hosts. In the first portion of my work, I develop a method to overcome the hurdles in characterizing and utilizing TCSs as biosensors. Through the framework of synthetic biology, I demonstrate that the two largest families of RR DNA binding domains (DBDs) can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. In collaboration with Kristina Daeffler, we exploit this plasticity to eliminate cross-regulation and in collaboration with Brian Landry, we un-silence a gram-negative TCS in a gram-positive host and engineer a sensor with over 1,300-fold activation. In collaboration with Kathryn Brink, we also apply DBD swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a novel pH sensor. In the second portion of my work, I demonstrate a method for identifying inhibitors of a virulence regulating TCS. This work focuses on the methicillin-resistant Staphylococcus aureus (MRSA) virulence regulating TCS SaeRS. I first heterologously express saeRS in Bacillus subtilis to remove the native, confounding regulation of the TCS and its output promoter. I then screen heterologous SaeRS against a diverse 1,593 small molecule library to identify inhibitors of its signaling. A lead compound emerged from the screen as an effective inhibitor of SaeRS signaling, leading to decreased exoprotein secretion and virulence from treated MRSA similar to MRSA lacking saeRS. My work described herein should accelerate 1) fundamental TCS studies and the engineering of a large family of biosensors with diverse applications and 2) the discovery of new anti-virulence compounds.
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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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    Identifying ligands for bacterial sensors
    (2020-10-06) Tabor, Jeffrey J.; Schmidl, Sebastian; Sheth, Ravi; Ekness, Felix; Landry, Brian; Dyuglyarov, Nikola; Rice University; United States Patent and Trademark Office
    Methods to create two component signal transduction systems by replace the DNA binding domains and output promoters in bacteria are described.
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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.