title: Transplantation of prokaryotic two-component signaling pathways into mammalian cells.
abstract: Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hansen
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland.
authors.forename: Erik
authors.initials: E
authors.lastname: Mailand
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland.
authors.forename: Krishna Kumar
authors.initials: KK
authors.lastname: Swaminathan
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland.
authors.forename: Joerg
authors.initials: J
authors.lastname: Schreiber
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland.
authors.forename: Bartolomeo
authors.initials: B
authors.lastname: Angelici
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich (ETH Zurich), CH-4058 Basel, Switzerland kobi.benenson@bsse.ethz.ch.
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Benenson
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D000071677:Histidine Kinase; D006801:Humans; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase
keywords: biological computing; synthetic biology; two-component signaling