title: Systematic transfer of prokaryotic sensors and circuits to mammalian cells.
abstract: Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10(5) members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Brynne C
authors.initials: BC
authors.lastname: Stanton
authors.forename: Velia
authors.initials: V
authors.lastname: Siciliano
authors.forename: Amar
authors.initials: A
authors.lastname: Ghodasara
authors.forename: Liliana
authors.initials: L
authors.lastname: Wroblewska
authors.forename: Kevin
authors.initials: K
authors.lastname: Clancy
authors.forename: Axel C
authors.initials: AC
authors.lastname: Trefzer
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Ron
authors.initials: R
authors.lastname: Weiss
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D010696:Phloroglucinol / Q000031:analogs & derivatives; D011401:Promoter Regions, Genetic / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; C470187:TetR(B) protein, E coli; C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol
keywords: 2,4-diacetylphloroglucinol (DAPG); eukaryote; inducible system; mammalian synthetic biology; systems biology