title: Synthetic biosensors for precise gene control and real-time monitoring of metabolites.
abstract: Characterization and standardization of inducible transcriptional regulators has transformed how scientists approach biology by allowing precise and tunable control of gene expression. Despite their utility, only a handful of well-characterized regulators exist, limiting the complexity of engineered biological systems. We apply a characterization pipeline to four genetically encoded sensors that respond to acrylate, glucarate, erythromycin and naringenin. We evaluate how the concentration of the inducing chemical relates to protein expression, how the extent of induction affects protein expression kinetics, and how the activation behavior of single cells relates to ensemble measurements. We show that activation of each sensor is orthogonal to the other sensors, and to other common inducible systems. We demonstrate independent control of three fluorescent proteins in a single cell, chemically defining eight unique transcriptional states. To demonstrate biosensor utility in metabolic engineering, we apply the glucarate biosensor to monitor product formation in a heterologous glucarate biosynthesis pathway and identify superior enzyme variants. Doubling the number of well-characterized inducible systems makes more complex synthetic biological circuits accessible. Characterizing sensors that transduce the intracellular concentration of valuable metabolites into fluorescent readouts enables high-throughput screening of biological catalysts and alleviates the primary bottleneck of the metabolic engineering design-build-test cycle.
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02143, USA.
authors.forename: Jameson K
authors.initials: JK
authors.lastname: Rogers
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Guzman
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Noah D
authors.initials: ND
authors.lastname: Taylor
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Srivatsan
authors.initials: S
authors.lastname: Raman
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA.
authors.forename: Kelley
authors.initials: K
authors.lastname: Anderson
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA gchurch@genetics.med.harvard.edu.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D005786:Gene Expression Regulation; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D060847:Metabolic Engineering; D064688:Metabolic Flux Analysis; D008957:Models, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D008164:Luminescent Proteins; D014157:Transcription Factors