Browsing by Author "Ellington, Andrew D."

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    Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes
    (Elsevier, 2025) Kar, Shaunak; Gardner, Elizabeth C.; Javanmardi, Kamyab; Boutz, Daniel R.; Shroff, Raghav; Horton, Andrew P.; Segall-Shapiro, Thomas H.; Ellington, Andrew D.; Gollihar, Jimmy; Bioengineering
    T7 RNA polymerase (RNAP) has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5′ methyl guanosine caps on T7 RNAP-derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.
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    A Humanized CB1R Yeast Biosensor Enables Facile Screening of Cannabinoid Compounds
    (MDPI, 2024) Mulvihill, Colleen J.; Lutgens, Joshua D.; Gollihar, Jimmy D.; Bachanová, Petra; Tramont, Caitlin; Marcotte, Edward M.; Ellington, Andrew D.; Gardner, Elizabeth C.; Bioengineering
    Yeast expression of human G-protein-coupled receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. Cannabinoid receptor type 1 (CB1R) is of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane modulates its performance. Using an engineered cannabinoid biosensor, we demonstrate that large libraries of synthetic cannabinoids and terpenes can be quickly screened to elucidate known and novel structure–activity relationships. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing therapeutic molecules.