title: Robust, tunable genetic memory from protein sequestration combined with positive feedback.
abstract: Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10(6)-fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Tatenda
authors.initials: T
authors.lastname: Shopera
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Henson
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Ng
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ng
authors.affiliation: Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA tsmoon@wustl.edu.
authors.forename: Tae Seok
authors.identifier: http://orcid.org/0000-0001-8373-9051
authors.initials: TS
authors.lastname: Moon
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D025461:Feedback, Physiological; D053263:Gene Regulatory Networks; D008957:Models, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods; D015534:Trans-Activators / Q000378:metabolism; D000069296:Type III Secretion Systems / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; C470740:ExsD protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D000069296:Type III Secretion Systems