title: Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems.
abstract: RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.
authors.affiliation: †School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.|‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Takahashi
authors.affiliation: †School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.
authors.forename: James
authors.initials: J
authors.lastname: Chappell
authors.affiliation: §Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Clarmyra A
authors.initials: CA
authors.lastname: Hayes
authors.affiliation: §Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Zachary Z
authors.initials: ZZ
authors.lastname: Sun
authors.affiliation: §Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Jongmin
authors.initials: J
authors.lastname: Kim
authors.affiliation: ‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.|§Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Vipul
authors.initials: V
authors.lastname: Singhal
authors.affiliation: ‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.|∥Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Texas 77030, United States.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Spring
authors.affiliation: ‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.|⊥Institute for Research in Immunology and Cancer (IRIC), Université de Montreal, Montreal, Quebec H3T 1J4, Canada.
authors.forename: Shaima
authors.initials: S
authors.lastname: Al-Khabouri
authors.affiliation: ‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.|∇Department of BioEngineering, University of Illinois, Chicago, Illinois 60607, United States.|#Department of Computer Science, Georgetown University, Washington, DC 20057, United States.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Fall
authors.affiliation: ○School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
authors.affiliation: ‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.|§Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Murray
authors.affiliation: †School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.|‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.
authors.forename: Julius B
authors.initials: JB
authors.lastname: Lucks
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D053263:Gene Regulatory Networks / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D014176:Protein Biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D012313:RNA
keywords: RNA genetic circuits; RNA synthetic biology; TX-TL; cell-free; response time