In vitro implementation of robust gene regulation in a synthetic biomolecular integral controller

Deepak K Agrawal,Ryan Marshall,Vincent Noireaux,Eduardo D Sontag

doi:10.1038/s41467-019-13626-z

Abstract

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Motivated by the need to tune the dynamics and improve the robustness of gene circuits, biological engineers have proposed various designs that mimic natural molecular feedback control mechanisms. However, practical and predictable implementations have proved challenging because of the complexity of synthesis and analysis of complex biomolecular networks. Here, we analyze and experimentally validate a synthetic biomolecular controller executed in vitro. The controller ensures that gene expression rate tracks an externally imposed reference level, and achieves this goal even in the presence of certain kinds of disturbances. Our design relies upon an analog of the well-known principle of integral feedback in control theory. We implement the controller in an Escherichia coli cell-free transcription-translation system, which allows rapid prototyping and implementation. Modeling and theory guide experimental implementation with well-defined operational predictability.

Highlights

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties
A phenomenon in which physiological variables are continuously monitored and adjusted so as to maintain a desired equilibrium value which is defined by a set point, in the presence of biological uncertainties that may perturb the natural state of the system[8,9,10]
We demonstrate that the output of the controller, which is realized using the TXTL toolbox, tracks the reference signal, meaning in this paper that it is linearly proportional to the input; we show that this happens for a large dynamic range of inputs

Summary

Introduction

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Inspired by these ideas from engineering and control theory, there have been several recent designs[13,14,15,16] and implementations[17,18,19] of biomolecular integral feedback controllers While these implementations provided a general framework to robustly regulate in vivo biological processes, an alternative implementation is desirable, in order to improve the robustness of synthetic biological processes and the cell-free reaction platform. Our results demonstrate that our synthetic biomolecular controller is capable of regulating the gene expression rate robustly in an E. coli TXTL toolbox We anticipate that such an approach could be useful for diagnostics applications[32], for constructing dynamical systems in vitro[33] or for programming synthetic cell systems[34,35]

Methods

Results

Conclusion