Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks

Synthetic biology aims to control and reprogram signal processing pathways within living cells so as to realize repurposed, beneficial applications. Here we report the design and construction of a set of modular and gain-tunable genetic amplifiers in Escherichia coli capable of amplifying a transcriptional signal with wide tunable-gain control in cascaded gene networks. The devices are engineered using orthogonal genetic components (hrpRS, hrpV and PhrpL) from the hrp (hypersensitive response and pathogenicity) gene regulatory network in Pseudomonas syringae. The amplifiers can linearly scale up to 21-fold the transcriptional input with a large output dynamic range, yet not introducing significant time delay or significant noise during signal amplification. The set of genetic amplifiers achieves different gains and input dynamic ranges by varying the expression levels of the underlying ligand-free activator proteins in the device. As their electronic counterparts, these engineered transcriptional amplifiers can act as fundamental building blocks in the design of biological systems by predictably and dynamically modulating transcriptional signal flows to implement advanced intra- and extra-cellular control functions.

[1]  J. Keasling,et al.  Engineering dynamic pathway regulation using stress-response promoters , 2013, Nature Biotechnology.

[2]  Rahul Sarpeshkar,et al.  Synthetic analog computation in living cells , 2013, Nature.

[3]  Drew Endy,et al.  Amplifying Genetic Logic Gates , 2013, Science.

[4]  M. Andersen,et al.  Ultrasensitive response motifs: basic amplifiers in molecular signalling networks , 2013, Open Biology.

[5]  Mauricio Barahona,et al.  A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals , 2013, Biosensors & bioelectronics.

[6]  Christopher A. Voigt,et al.  Genetic programs constructed from layered logic gates in single cells , 2012, Nature.

[7]  Eric Klavins,et al.  Fine-tuning gene networks using simple sequence repeats , 2012, Proceedings of the National Academy of Sciences.

[8]  Baojun Wang,et al.  Customizing cell signaling using engineered genetic logic circuits. , 2012, Trends in microbiology.

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

[10]  James J. Collins,et al.  Genetic switchboard for synthetic biology applications , 2012, Proceedings of the National Academy of Sciences.

[11]  J. Keasling,et al.  Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids , 2012, Nature Biotechnology.

[12]  D. Pincus,et al.  In silico feedback for in vivo regulation of a gene expression circuit , 2011, Nature Biotechnology.

[13]  Martin Fussenegger,et al.  Emerging biomedical applications of synthetic biology , 2011, Nature Reviews Genetics.

[14]  Baojun Wang,et al.  Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology , 2011, Nature communications.

[15]  Martin Buck,et al.  Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity , 2011, Nature communications.

[16]  Martin Fussenegger,et al.  Self-sufficient control of urate homeostasis in mice by a synthetic circuit , 2010, Nature Biotechnology.

[17]  Kang Wu,et al.  A modular positive feedback-based gene amplifier , 2010, Journal of biological engineering.

[18]  L. Serrano,et al.  Engineering Signal Transduction Pathways , 2010, Cell.

[19]  Domitilla Del Vecchio,et al.  Design and Characterization of a Three-terminal Transcriptional Device Through Polymerase Per Second , 2009, IEEE Transactions on NanoBioscience.

[20]  Christopher A. Voigt,et al.  Spatiotemporal Control of Cell Signalling Using A Light-Switchable Protein Interaction , 2009, Nature.

[21]  C. Townsend,et al.  An externally tunable bacterial band-pass filter , 2009, Proceedings of the National Academy of Sciences.

[22]  M. Bennett,et al.  A fast, robust, and tunable synthetic gene oscillator , 2008, Nature.

[23]  Drew Endy,et al.  Engineering BioBrick vectors from BioBrick parts , 2008, Journal of Biological Engineering.

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

[25]  J. Gallivan,et al.  Guiding bacteria with small molecules and RNA. , 2007, Journal of the American Chemical Society.

[26]  D. Bose,et al.  A second paradigm for gene activation in bacteria. , 2006, Biochemical Society transactions.

[27]  U. Bonas,et al.  Who comes first? How plant pathogenic bacteria orchestrate type III secretion. , 2006, Current opinion in microbiology.

[28]  G. Stephanopoulos,et al.  Tuning genetic control through promoter engineering. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Weiss,et al.  Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S.W. Davies,et al.  A genetic circuit amplifier: design and simulation , 2003, IEEE Transactions on NanoBioscience.

[31]  S. He,et al.  Type III protein secretion in Pseudomonas syringae. , 2003, Microbes and infection.

[32]  S. Hutcheson,et al.  Enhancer-Binding Proteins HrpR and HrpS Interact To Regulate hrp-Encoded Type III Protein Secretion inPseudomonas syringae Strains , 2001, Journal of bacteriology.

[33]  D. Fouts,et al.  Pseudomonas syringae Hrp type III secretion system and effector proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  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.

[35]  A. Collmer,et al.  Negative Regulation of hrp Genes inPseudomonas syringae by HrpV , 1998, Journal of bacteriology.

[36]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[37]  M. Dubow,et al.  An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria , 1995, Journal of bacteriology.

[38]  P.E. Allen,et al.  Operational amplifiers and linear integrated circuits , 1983, Proceedings of the IEEE.

[39]  H.T. Friis,et al.  Noise Figures of Radio Receivers , 1944, Proceedings of the IRE.

[40]  Douglas B. Kell,et al.  System Modeling in Cell Biology From Concepts to Nuts and Bolts , 2005 .

[41]  Christin Wirth.  The Essential Physics of Medical Imaging , 2003, European Journal of Nuclear Medicine and Molecular Imaging.