Browsing by Author "Daeffler, Kristina N-M"

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    Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation
    (EMBO Press, 2017) Daeffler, Kristina N-M; Galley, Jeffrey D.; Sheth, Ravi U.; Ortiz-Velez, Laura C.; Bibb, Christopher O.; Shroyer, Noah F.; Britton, Robert A.; Tabor, Jeffrey J.; Bioengineering; Biosciences
    There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two?component systems from marine Shewanella species, and validate them in laboratory Escherichiaﾠcoli. Then, we port these sensors into a gut?adapted probiotic E.ﾠcoli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.
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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.; Bioengineering; Biosciences
    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.