Robust, tunable genetic memory from protein sequestration combined with positive feedback

Nucleic Acids Res. 2015 Oct 15;43(18):9086-94. doi: 10.1093/nar/gkv936. Epub 2015 Sep 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism
  • Escherichia coli / genetics
  • Feedback, Physiological
  • Gene Regulatory Networks*
  • Models, Genetic
  • Pseudomonas aeruginosa / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / metabolism
  • Synthetic Biology / methods
  • Trans-Activators / metabolism
  • Type III Secretion Systems / genetics


  • Bacterial Proteins
  • ExsA protein, Pseudomonas aeruginosa
  • ExsD protein, Pseudomonas aeruginosa
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Trans-Activators
  • Type III Secretion Systems