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

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 synthetic 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 first synthetic biomolecular controller executed in vitro. The controller is based on the interaction between a sigma and an anti-sigma factor, which ensures that gene expression tracks an externally imposed reference level, and achieves this goal even in the presence 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 (TXTL) system, a platform that allows rapid prototyping and implementation. Modeling and theory guide experimental implementation of the controller with well-defined operational predictability.

[1]  Vincent Noireaux,et al.  Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell , 2016, Artificial Life.

[2]  Ankit Gupta,et al.  Antithetic Integral Feedback Ensures Robust Perfect Adaptation in Noisy Biomolecular Networks. , 2014, Cell systems.

[3]  D. Vecchio,et al.  Biomolecular Feedback Systems , 2014 .

[4]  K Oishi,et al.  Biomolecular implementation of linear I/O systems. , 2011, IET systems biology.

[5]  M. Hoagland,et al.  Feedback Systems An Introduction for Scientists and Engineers SECOND EDITION , 2015 .

[6]  Vincent Noireaux,et al.  A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs. , 2018, Methods.

[7]  Mustafa Khammash,et al.  A synthetic integral feedback controller for robust tunable regulation in bacteria , 2017, bioRxiv.

[8]  R. F. Wang,et al.  Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. , 1991, Gene.

[9]  Jay D. Keasling,et al.  A model for improving microbial biofuel production using a synthetic feedback loop , 2010, Systems and Synthetic Biology.

[10]  Vincent Noireaux,et al.  Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. , 2014, Biochimie.

[11]  Eduardo Sontag,et al.  Modular cell biology: retroactivity and insulation , 2008, Molecular systems biology.

[12]  Domitilla Del Vecchio,et al.  A quasi-integral controller for adaptation of genetic modules to variable ribosome demand , 2018, Nature Communications.

[13]  Jeffrey D Varner,et al.  Generating Effective Models and Parameters for RNA Genetic Circuits. , 2015, ACS synthetic biology.

[14]  J. Doyle,et al.  Robust perfect adaptation in bacterial chemotaxis through integral feedback control. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Khammash,et al.  Antithetic Integral Feedback Ensures Robust Perfect Adaptation in Noisy Biomolecular Networks. , 2016, Cell systems.

[16]  Vincent Noireaux,et al.  Programmable on-chip DNA compartments as artificial cells , 2014, Science.

[17]  P. Swain,et al.  Stochastic Gene Expression in a Single Cell , 2002, Science.

[18]  M. Khammash,et al.  A universal biomolecular integral feedback controller for robust perfect adaptation , 2019, Nature.

[19]  Xun Tang,et al.  Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression. , 2018, ACS synthetic biology.

[20]  A. Arkin,et al.  Contextualizing context for synthetic biology – identifying causes of failure of synthetic biological systems , 2012, Biotechnology journal.

[21]  R. Murray,et al.  Gene circuit performance characterization and resource usage in a cell-free "breadboard". , 2014, ACS synthetic biology.

[22]  Michael L. Dustin,et al.  Feedback control of regulatory T cell homeostasis by dendritic cells in vivo , 2009, The Journal of experimental medicine.

[23]  Domitilla Del Vecchio,et al.  Realizing “integral control” in living cells: How to overcome leaky integration due to dilution? , 2017, bioRxiv.

[24]  W. Lim,et al.  Defining Network Topologies that Can Achieve Biochemical Adaptation , 2009, Cell.

[25]  Andreas W. K. Harris,et al.  Synthetic negative feedback circuits using engineered small RNAs , 2017, bioRxiv.

[26]  Eduardo Sontag,et al.  Untangling the wires: A strategy to trace functional interactions in signaling and gene networks , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Franco Blanchini,et al.  Molecular Titration Promotes Oscillations and Bistability in Minimal Network Models with Monomeric Regulators. , 2016, ACS synthetic biology.

[28]  R. Ramphal,et al.  Identification and Functional Characterization of flgM, a Gene Encoding the Anti-Sigma 28 Factor in Pseudomonas aeruginosa , 2002, Journal of bacteriology.

[29]  J. Collins,et al.  Synthetic biology devices for in vitro and in vivo diagnostics , 2015, Proceedings of the National Academy of Sciences.

[30]  R. Zimmer,et al.  Experiment and mathematical modeling of gene expression dynamics in a cell-free system. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[31]  S. Leibler,et al.  Robustness in simple biochemical networks , 1997, Nature.

[32]  Vincent Noireaux,et al.  The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. , 2016, ACS synthetic biology.

[33]  Eduardo D. Sontag,et al.  Mathematical Control Theory: Deterministic Finite Dimensional Systems , 1990 .

[34]  Julius B. Lucks,et al.  Distinct timescales of RNA regulators enable the construction of a genetic pulse generator , 2018, bioRxiv.

[35]  Richard M. Murray,et al.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology , 2013, Journal of visualized experiments : JoVE.

[36]  Christopher A. Voigt,et al.  Engineered promoters enable constant gene expression at any copy number in bacteria , 2018, Nature Biotechnology.

[37]  Zhen Xie,et al.  Molecular Systems Biology Peer Review Process File Synthetic Incoherent Feed-forward Circuits Show Adaptation to the Amount of Their Genetic Template. Transaction Report , 2022 .

[38]  Vincent Noireaux,et al.  Cell-sized mechanosensitive and biosensing compartment programmed with DNA. , 2017, Chemical communications.

[39]  Zachary Z. Sun,et al.  Characterizing and prototyping genetic networks with cell-free transcription-translation reactions. , 2015, Methods.

[40]  Lorenzo Marconi,et al.  Internal Models in Control, Biology and Neuroscience , 2018, 2018 IEEE Conference on Decision and Control (CDC).

[41]  U. Alon,et al.  Robustness in bacterial chemotaxis , 2022 .

[42]  Hana El-Samad,et al.  Design and analysis of a Proportional-Integral-Derivative controller with biological molecules , 2018, bioRxiv.

[43]  Mustafa Khammash,et al.  Design of a synthetic integral feedback circuit: dynamic analysis and DNA implementation , 2016, ACS synthetic biology.

[44]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[45]  C. Rao,et al.  Control, exploitation and tolerance of intracellular noise , 2002, Nature.

[46]  Julius B. Lucks,et al.  Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator , 2016, bioRxiv.