Sequestration-based bistability enables tuning of the switching boundaries and design of a latch

Natural biological systems have evolved a diverse array of switches to realize their strategies for environmental response and development. Emerging applications of synthetic biology have begun to exploit such switches to achieve increasingly sophisticated designed behaviors. However, not all switch architectures allow facile design of the switching and memory properties. Furthermore, not all designs are built from components for which large families of variants exist, a requirement for building many orthogonal switch variants. Therefore, there is a critical need from genetic engineers for scalable strategies that yield custom bistable switches. Here, we use a sigma factor and its cognate anti‐sigma factor to experimentally verify that ultrasensitivity from sequestration combined with positive feedback is sufficient to build a bistable switch. We show that sequestration allows us to predictably tune the switching boundaries, and we can easily tune our switch to function as a set–reset latch that can be toggled between two states by a pulse of inducer input.

[1]  T. Donohue,et al.  ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene , 1995, Journal of bacteriology.

[2]  J. Ferrell.  Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs. , 1996, Trends in biochemical sciences.

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

[4]  J. Keasling,et al.  Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. , 2001, Microbiology.

[5]  J. Newman,et al.  The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor. , 2001, Journal of molecular biology.

[6]  J. Helmann,et al.  The -10 region is a key promoter specificity determinant for the Bacillus subtilis extracytoplasmic-function sigma factors sigma(X) and sigma(W). , 2001, Journal of bacteriology.

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

[8]  J. Helmann,et al.  The −10 Region Is a Key Promoter Specificity Determinant for the Bacillus subtilis Extracytoplasmic-Function ς Factors ςX and ςW , 2001 .

[9]  J. Ferrell.  Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. , 2002, Current opinion in cell biology.

[10]  A. Ninfa,et al.  Development of Genetic Circuitry Exhibiting Toggle Switch or Oscillatory Behavior in Escherichia coli , 2003, Cell.

[11]  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.

[12]  V. Hakim,et al.  Design of genetic networks with specified functions by evolution in silico. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W. Schumann,et al.  The Bacillus subtilisσW anti‐sigma factor RsiW is degraded by intramembrane proteolysis through YluC , 2004, Molecular microbiology.

[14]  Ertugrul M. Ozbudak,et al.  Multistability in the lactose utilization network of Escherichia coli , 2004, Nature.

[15]  Martin Fussenegger,et al.  Hysteresis in a synthetic mammalian gene network. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Oudenaarden,et al.  Enhancement of cellular memory by reducing stochastic transitions , 2005, Nature.

[17]  W. Lane,et al.  The Structural Basis for Promoter −35 Element Recognition by the Group IV σ Factors , 2006, PLoS biology.

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

[19]  W. Schumann,et al.  Involvement of Clp protease activity in modulating the Bacillus subtilisσW stress response , 2006, Molecular microbiology.

[20]  James E. Ferrell,et al.  Substrate Competition as a Source of Ultrasensitivity in the Inactivation of Wee1 , 2007, Cell.

[21]  S. Elledge,et al.  Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC , 2007, Nature Methods.

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

[23]  Nicolas E. Buchler,et al.  Molecular titration and ultrasensitivity in regulatory networks. , 2008, Journal of molecular biology.

[24]  C. Bashor,et al.  References and Notes Supporting Online Material Using Engineered Scaffold Interactions to Reshape Map Kinase Pathway Signaling Dynamics , 2022 .

[25]  Timothy S. Ham,et al.  Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory , 2008, PloS one.

[26]  T. Hwa,et al.  Growth Rate-Dependent Global Effects on Gene Expression in Bacteria , 2009, Cell.

[27]  Nicolas E. Buchler,et al.  Protein sequestration generates a flexible ultrasensitive response in a genetic network , 2009, Molecular systems biology.

[28]  L. You,et al.  Emergent bistability by a growth-modulating positive feedback circuit. , 2009, Nature chemical biology.

[29]  Luke E. Ulrich,et al.  The third pillar of bacterial signal transduction: classification of the extracytoplasmic function (ECF) σ factor protein family , 2009, Molecular microbiology.

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

[31]  Gábor Balázsi,et al.  The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response , 2010, Physical biology.

[32]  Pamela A. Silver,et al.  Making Cellular Memories , 2010, Cell.

[33]  Haisu Ma,et al.  Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch , 2010, Molecular systems biology.

[34]  M. Elowitz,et al.  A synthetic three-color scaffold for monitoring genetic regulation and noise , 2010, Journal of biological engineering.

[35]  M. West,et al.  Image segmentation and dynamic lineage analysis in single‐cell fluorescence microscopy , 2009, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[36]  J. Ferrell,et al.  Ultrasensitivity in the Regulation of Cdc25C by Cdk1. , 2011, Molecular cell.

[37]  Oleg A. Igoshin,et al.  Non-transcriptional regulatory processes shape transcriptional network dynamics , 2011, Nature Reviews Microbiology.

[38]  Santhosh Palani,et al.  Synthetic conversion of a graded receptor signal into a tunable, reversible switch , 2011, Molecular systems biology.

[39]  Gábor Balázsi,et al.  Mapping the Environmental Fitness Landscape of a Synthetic Gene Circuit , 2012, PLoS Comput. Biol..

[40]  D. Endy,et al.  Rewritable digital data storage in live cells via engineered control of recombination directionality , 2012, Proceedings of the National Academy of Sciences.

[41]  Narendra Maheshri,et al.  A regulatory role for repeated decoy transcription factor binding sites in target gene expression , 2012, Molecular systems biology.

[42]  K. Prehoda,et al.  Ultrasensitive synthetic protein regulatory networks using mixed decoys. , 2012, ACS synthetic biology.