Synthetic conversion of a graded receptor signal into a tunable, reversible switch

The ability to engineer an all‐or‐none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network ‘switches’ have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (nH>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady‐state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal‐transduction pathways.

[1]  J. Hasty,et al.  Noise-based switches and amplifiers for gene expression. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Farren J. Isaacs,et al.  Prediction and measurement of an autoregulatory genetic module , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  James J. Collins,et al.  A Tunable Genetic Switch Based on RNAi and Repressor Proteins for Regulating Gene Expression in Mammalian Cells , 2007, Cell.

[4]  Santhosh Palani,et al.  Positive receptor feedback during lineage commitment can generate ultrasensitivity to ligand and confer robustness to a bistable switch. , 2008, Biophysical journal.

[5]  M. Mok,et al.  CYTOKININ METABOLISM AND ACTION. , 2003, Annual review of plant physiology and plant molecular biology.

[6]  Michel Sadelain,et al.  The promise and potential pitfalls of chimeric antigen receptors. , 2009, Current Opinion in Immunology.

[7]  G. Church,et al.  Synthetic Gene Networks That Count , 2009, Science.

[8]  R. Weiss,et al.  A universal RNAi-based logic evaluator that operates in mammalian cells , 2007, Nature Biotechnology.

[9]  R. Deschenes,et al.  Saccharomyces cerevisiae Histidine Phosphotransferase Ypd1p Shuttles between the Nucleus and Cytoplasm for SLN1-Dependent Phosphorylation of Ssk1p and Skn7p , 2003, Eukaryotic Cell.

[10]  Kazuo Shinozaki,et al.  Identification of CRE1 as a cytokinin receptor from Arabidopsis , 2001, Nature.

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

[12]  Naama Barkai,et al.  Threshold responses to morphogen gradients by zero-order ultrasensitivity , 2005, Molecular systems biology.

[13]  Hannah H. Chang,et al.  Multistable and multistep dynamics in neutrophil differentiation , 2006, BMC Cell Biology.

[14]  Christopher A. Voigt,et al.  Environmental signal integration by a modular AND gate , 2007, Molecular systems biology.

[15]  J. Collins,et al.  Construction of a genetic toggle switch in Escherichia coli , 2000, Nature.

[16]  J E Ferrell,et al.  The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. , 1998, Science.

[17]  G. Stephanopoulos,et al.  Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? , 2009, Nature Reviews Microbiology.

[18]  R. Kwok.  Five hard truths for synthetic biology , 2010, Nature.

[19]  Shankar Mukherji,et al.  Synthetic biology: understanding biological design from synthetic circuits , 2009, Nature Reviews Genetics.

[20]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[21]  M. Fussenegger,et al.  An engineered epigenetic transgene switch in mammalian cells , 2004, Nature Biotechnology.

[22]  R. Weiss,et al.  Directed evolution of a genetic circuit , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  James E. Ferrell,et al.  The JNK Cascade as a Biochemical Switch in Mammalian Cells Ultrasensitive and All-or-None Responses , 2003, Current Biology.

[24]  E. Winfree,et al.  Construction of an in vitro bistable circuit from synthetic transcriptional switches , 2006, Molecular systems biology.

[25]  Wendell A Lim,et al.  Engineering synthetic signaling proteins with ultrasensitive input/output control , 2007, Nature Biotechnology.

[26]  B. Séraphin,et al.  Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion , 2001, The EMBO journal.

[27]  David A. Drubin,et al.  Rational design of memory in eukaryotic cells. , 2007, Genes & development.

[28]  R. Weiss,et al.  Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana , 2005, Nature Biotechnology.

[29]  M. Win,et al.  Higher-Order Cellular Information Processing with Synthetic RNA Devices , 2008, Science.