title: Creating Single-Copy Genetic Circuits.
abstract: Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use.
authors.affiliation: Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
authors.forename: Jeong Wook
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA.
authors.forename: Andras
authors.initials: A
authors.lastname: Gyorgy
authors.affiliation: Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
authors.forename: D Ewen
authors.initials: DE
authors.lastname: Cameron
authors.affiliation: Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
authors.forename: Nora
authors.initials: N
authors.lastname: Pyenson
authors.affiliation: Department of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.
authors.forename: Kyeong Rok
authors.initials: KR
authors.lastname: Choi
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Way
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
authors.affiliation: Department of Mechanical Engineering, MIT, Cambridge, MA 02139, USA.
authors.forename: Domitilla
authors.initials: D
authors.lastname: Del Vecchio
authors.affiliation: Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA; Harvard-MIT Program in Health Sciences and Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: jimjc@mit.edu.
authors.forename: James J
authors.initials: JJ
authors.lastname: Collins
mesh_terms: D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D013269:Stochastic Processes; D058615:Synthetic Biology / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D008164:Luminescent Proteins; C470187:TetR(B) protein, E coli