title: A general strategy to construct small molecule biosensors in eukaryotes.
abstract: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.
authors.affiliation: Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, United States.|Department of Genetics, Harvard Medical School, Boston, United States.
authors.forename: Justin
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, United States.|Howard Hughes Medical Institute, University of Washington, Seattle, United States.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Jester
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, United States.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Tinberg
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, United States.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, United States.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Mandell
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, United States.
authors.forename: Mauricio S
authors.initials: MS
authors.lastname: Antunes
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, United States.
authors.forename: Raj
authors.initials: R
authors.lastname: Chari
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, United States.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Morey
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, United States.
authors.forename: Xavier
authors.initials: X
authors.lastname: Rios
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, United States.
authors.forename: June I
authors.initials: JI
authors.lastname: Medford
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, United States.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, United States.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, United States.|Howard Hughes Medical Institute, University of Washington, Seattle, United States.|Department of Medicine, University of Washington, Seattle, United States.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.affiliation: Howard Hughes Medical Institute, University of Washington, Seattle, United States.|Department of Biochemistry, University of Washington, Seattle, United States.
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004077:Digoxin / Q000032:analysis; D056890:Eukaryota; D008967:Molecular Biology / Q000379:methods*; D011374:Progesterone / Q000032:analysis; D011485:Protein Binding; D055550:Protein Stability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D011374:Progesterone; D004077:Digoxin
keywords: <i>a. thaliana</i>; <i>s. cerevisiae</i>; CRISPR; biochemistry; biophysics; biosensors; human; metabolic engineering; structural biology