title: Multi-input regulation and logic with T7 promoters in cells and cell-free systems.
abstract: Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.
authors.affiliation: Graduate Program in Genome Science and Technology, University of Tennessee, Knoxville, Knoxville, Tennessee, United States of America ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors.forename: Sukanya
authors.initials: S
authors.lastname: Iyer
authors.forename: David K
authors.initials: DK
authors.lastname: Karig
authors.forename: S Elizabeth
authors.initials: SE
authors.lastname: Norred
authors.forename: Michael L
authors.initials: ML
authors.lastname: Simpson
authors.forename: Mitchel J
authors.initials: MJ
authors.lastname: Doktycz
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D004926:Escherichia coli; D053263:Gene Regulatory Networks / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein