Forward engineering of synthetic bio-logical and gates

The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms seen in digital electronic circuits. Examples are bistable switches, oscillators, and logic gates. The present work combines detailed mechanistic-kinetic models and stochastic simulation techniques as well as the techniques of in vivo molecular biology to study the potential of a synthetic, single promoter AND gate. This device is composed of elements of the tet, lac, and -phage promoters and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal. The quantitative behavior of the AND gate phenotype is studied both in numero and in vivo as a function of promoter topology. The model is constructed from kinetic data obtained from the literature and yields clearly defined ON/OFF logical behavior at realistic inducer concentrations. These behaviors are matched with observed in vivo data obtained through fluorescence-activated cell sorting. The effect of incomplete repression by weaker LacI repressor is also investigated and quantified. The simulation results, coupled with in vivo data, not only identify important design degrees of freedom, but also provide parameters that can be used to guide future synthetic designs using these common regulatory elements.

[1]  M. Elowitz,et al.  Combinatorial Synthesis of Genetic Networks , 2002, Science.

[2]  Vassilios Sotiropoulos,et al.  An adaptive time step scheme for a system of stochastic differential equations with multiple multiplicative noise: chemical Langevin equation, a proof of concept. , 2008, The Journal of chemical physics.

[3]  W. Hillen,et al.  Tetracycline analogs affecting binding to Tn10-Encoded Tet repressor trigger the same mechanism of induction. , 1996, Biochemistry.

[4]  M. Elowitz,et al.  A synthetic oscillatory network of transcriptional regulators , 2000, Nature.

[5]  W. Hillen,et al.  Thermodynamic analysis of tetracycline-mediated induction of Tet repressor by a quantitative methylation protection assay. , 1995, Analytical biochemistry.

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

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

[8]  M. Thomas Record,et al.  RNA Polymerase-Promoter Interactions: the Comings and Goings of RNA Polymerase , 1998, Journal of bacteriology.

[9]  Vassilios Sotiropoulos,et al.  SynBioSS: the synthetic biology modeling suite , 2008, Bioinform..

[10]  H. Salis,et al.  Computer-aided design of modular protein devices: Boolean AND gene activation , 2006, Physical biology.

[11]  E. Kanin,et al.  Holoenzyme switching and stochastic release of sigma factors from RNA polymerase in vivo. , 2005, Molecular cell.

[12]  A. Arkin,et al.  Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli cells. , 1998, Genetics.

[13]  Yiannis Kaznessis,et al.  Accurate hybrid stochastic simulation of a system of coupled chemical or biochemical reactions. , 2005, The Journal of chemical physics.

[14]  Vassilios Sotiropoulos,et al.  Multiscale Hy3S: Hybrid stochastic simulation for supercomputers , 2006, BMC Bioinformatics.

[15]  Yiannis N. Kaznessis,et al.  Models for synthetic biology , 2007, BMC Systems Biology.

[16]  R. Weiss,et al.  Advances in synthetic biology: on the path from prototypes to applications. , 2005, Current opinion in biotechnology.

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

[18]  J M Rosenberg,et al.  Kinetic studies of inducer binding to lac repressor.operator complex. , 1980, The Journal of biological chemistry.

[19]  Howard M. Salis,et al.  Numerical simulation of stochastic gene circuits , 2005, Comput. Chem. Eng..

[20]  P. V. von Hippel,et al.  Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA. , 1977, Biochemistry.

[21]  Jeff Hasty,et al.  Engineered gene circuits , 2002, Nature.

[22]  U. Alon,et al.  Detailed map of a cis-regulatory input function , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Jeffrey C Way,et al.  Designing biological systems. , 2007, Genes & development.

[24]  M Lanzer,et al.  Promoters largely determine the efficiency of repressor action. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[25]  H. Bujard,et al.  Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. , 1997, Nucleic acids research.

[26]  L. Serrano,et al.  Engineering stability in gene networks by autoregulation , 2000, Nature.

[27]  M. O'Neill.  Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization. , 1989, The Journal of biological chemistry.

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

[29]  M. Elowitz,et al.  Reconstruction of genetic circuits , 2005, Nature.

[30]  J. Chin,et al.  Cellular logic with orthogonal ribosomes. , 2005, Journal of the American Chemical Society.

[31]  Martin Fussenegger,et al.  BioLogic gates enable logical transcription control in mammalian cells , 2004, Biotechnology and bioengineering.

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

[33]  J. Collins,et al.  Programmable cells: interfacing natural and engineered gene networks. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Yiannis N Kaznessis,et al.  Optimization of a stochastically simulated gene network model via simulated annealing. , 2006, Biophysical journal.

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

[36]  Yiannis N Kaznessis,et al.  Model-driven designs of an oscillating gene network. , 2005, Biophysical journal.

[37]  M. Elowitz,et al.  Programming gene expression with combinatorial promoters , 2007, Molecular systems biology.

[38]  Vassilios Sotiropoulos,et al.  Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes , 2007, BMC Systems Biology.

[39]  U. Alon,et al.  Plasticity of the cis-Regulatory Input Function of a Gene , 2006, PLoS biology.

[40]  Jeffrey W. Smith,et al.  Stochastic Gene Expression in a Single Cell , .