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 106-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.

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

“…Until recently, most genetic circuits have been built using protein regulators. Though complex genetic circuits have been constructed using proteins, including a toggle switch ( 2 ), a repressilator ( 3 ), layered logic gates ( 4 ) and memory devices ( 5 – 7 ), there are inherent limitations of protein regulators that have stymied efforts to build complex and robust circuits ( 8 ). RNA regulators are emerging as simple, tunable and orthogonal genetic parts that can overcome many of the limitations of protein regulators.…”

Dynamics of sequestration-based gene regulatory cascades

“…It is worth noting that the steady-state free ExsA concentrations used in this work are model-derived values that were obtained by considering experimentally measured parameters, typical parameters for bacteria, and physiologically relevant bacterial protein concentration ranges (13,15,33,35–39). Thus, the steady-state free ExsA concentrations are just reasonably estimated values, not accurate ones.…”

Splitting nodes and linking channels: A method for assembling biocircuits from stochastic elementary units

“…In [41] an ultrasensitive synthetic transcriptional cascade was constructed where it was noted that a proper matching of the kinetic rates of the cascade's elements are crucial for a clear separation between the ON and OFF states. The design and the construction of a noise-tolerant ultrasensitive biocircuit was reported in [42]. Ultrasensitivity can be achieved by more than one network topology.…”