The Sixth q-bio Summer School: Synthetic Biology

In this theme, students will learn how to use computational modeling tools for design and evaluation of synthetic gene networks. We will review basic modeling approaches including Boolean networks, mass action kinetics, stochastic simulation algorithms and provide hands-on training sessions on using corresponding software tools. Then we will apply these tools to the design and analysis of basic elements of synthetic circuits such as positive and feedback loops, oscillators, toggle switches, logical gates, quorum-sensing circuits, enzymatic machinery. Issues of stochasticity, cell-to-cell variability, robustness and parameter sensitivity will be addressed in depth in regards to evaluation of synthetic circuit performance.

Lecturers

• Matt Bennett, Rice University
• Tim Elston, University of North Carolina
• Jeff M. Hasty, University of California, San Diego
• Lev S. Tsimring, University of California, San Diego
• Ron Weiss, MIT

Project Mentors

• Will Mather
• Mike Ferry
• Jangir Selimkhanov
• Brooks Taylor
• Phillip Samayoa

Topics

• Biochemical Reaction Kinetics: Elementary Reactions, Law of Mass Action,

Generalized Mass Action, Chemical Equilibrium, Enzyme Kinetics, Stochastic Reaction Kinetics

• Promoter Dynamics: Repressor-Operator Binding, Alternative Reaction Paths,

Cooperative Transcription Factor Binding, Synergism in RNA Polymerase Binding, DNA looping

• Simple cis-regulatory Systems: Plac promoter, Gal1 promoter
• Complex cis-regulatory Systems: Hierarchical Representations, cis-regulatory

Computational Logic

• Positive and Negative Feedback
• Bistable Switches: Lambda and the Engineered Toggle Switch
• Logic gates and pulse generators
• Genetic Oscillators: positive-negative feedback, time-delayed negative feedback,

segmentation clock

• Signaling cascades and regulatory motifs
• Sources of gene expression noise: translational bursting, transcriptional bursting,

transcription factor fluctuations

• Noise in gene networks: feedback systems - reduction by negative feedback and

amplification by positive feedback

• Noise-induced transitions and bistability

Suggested Reading

[1] Gibson, D. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52 (2010).

[2] Hasty, J., McMillen, D. & Collins, J. J. Engineered gene circuits. Nature 420, 224–30 (2002).

[3] Sprinzak, D. & Elowitz, M. B. Reconstruction of genetic circuits. Nature 438, 443–8 (2005).

[4] Endy, D. Foundations for engineering biology. Nature 438, 449–53 (2005).

[5] Ellis, T., Wang, X. & Collins, J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature biotechnology 27, 465 (2009).

[6] Kobayashi, H. et al. Programmable cells: interfacing natural and engineered gene networks. Proc. Natl. Acad. Sci. U. S. A. 101, 8414–9 (2004).

[7] You, L., Cox, I., R. S.,Weiss, R. & Arnold, F. H. Programmed population control by cell-cell communication and regulated killing. Nature 428, 868–71 (2004).

[8] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–4 (2005).

[9] Mukherji, S.&Van Oudenaarden, A. Synthetic biology: understanding biological design from synthetic circuits. Nature Reviews Genetics 10, 859–871 (2009).

[10] Grilly, C., Stricker, J., Pang,W., Bennett, M.&Hasty, J. A synthetic gene network for tuning protein degradation in saccharomyces cerevisiae. Molecular systems biology 3 (2007).

[11] Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–42 (2000).

[12] Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–8 (2000).

[13] Lu, T. & Collins, J. Dispersing biofilms with engineered enzymatic bacteriophage. Proceedings of the National Academy of Sciences 104, 11197 (2007).

[14] Friedland, A. et al. Synthetic gene networks that count. Science 324, 1199 (2009).

[15] Danino, T., Mondragon-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010).

[16] Tamsir, A., Tabor, J. & Voigt, C. Robust multicellular computing using genetically encoded nor gates and chemical/wires/’. Nature 469, 212–215 (2010).

[17] Tabor, J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).

[18] Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–9 (2008).

[19] Mondragon-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Entrainment of a population of synthetic genetic oscillators. Science 333 (2011).

[20] Tigges, M., Marquez-Lago, T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009).

[21] Westinghouse, G. System of electrical distribution. U.S. Patent No. 373,035 (1887).

[22] Lewandowski, W., Azoubib, J. & Klepczynski, W. Gps: Primary tool for time transfer. Proceedings of the IEEE 87, 163–172 (1999).

[23] Vladimirov, A., Kozyreff, G. & Mandel, P. Synchronization of weakly stable oscillators and semiconductor laser arrays. EPL (Europhysics Letters) 61, 613 (2003).

[24] Gast, T. Sensors with oscillating elements. Journal of Physics E: Scientific Instruments 18, 783 (1985).

[25] Ozbudak, E. M., Thattai, M., Kurtser, I., Grossman, A. D. & van Oudenaarden, A. Regulation of noise in the expression of a single gene. Nat. Genet. 31, 69–73 (2002).

[26] Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–6 (2002).

[27] Golding, I., Paulsson, J., Zawilski, S. & Cox, E. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

[28] Blake, W. et al. Phenotypic consequences of promoter-mediated transcriptional noise. Molecular cell 24, 853–865 (2006).

[29] Austin, D. et al. Gene network shaping of inherent noise spectra. Nature 439, 608–611 (2006).

[30] Ferry, M., Razinkov, I. & Hasty, J. Microfluidics for synthetic biology from design to execution. Methods in enzymology 497, 295 (2011).

[31] Messner, K. & Imlay, J. The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of escherichia coli. Journal of Biological Chemistry 274, 10119 (1999).

[32] Bose, J. L. et al. Bioluminescence in vibrio fischeri is controlled by the redoxresponsive regulator arca. Molecular Microbiology 65, 538–553 (2007).

[33] Georgellis, D., Kwon, O. & Lin, E. Quinones as the redox signal for the arc two-component system of bacteria. Science 292, 2314 (2001).

[34] Seaver, L. & Imlay, J. Hydrogen peroxide fluxes and compartmentalization inside growing escherichia coli. Journal of bacteriology 183, 7182 (2001).

[35] Fridovich, I. The biology of oxygen radicals. Science 201, 875 (1978).

[36] McCord, J. & Fridovich, I. Superoxide dismutase. Journal of Biological Chemistry 244, 6049 (1969).

[37] Berg, J., Tymoczko, J. L. & Stryer, L. Biochemistry (W.H. Freeman, 2006).

[38] Remington, S. Fluorescent proteins: maturation, photochemistry and photophysics. Current opinion in structural biology 16, 714–721 (2006).

[39] Kelner, M., Bagnell, R. & Welch, K. Thioureas react with superoxide radicals to yield a sulfhydryl compound. explanation for protective effect against paraquat. Journal of Biological Chemistry 265, 1306 (1990).

[40] Touati, D., Jacques, M., Tardat, B., Bouchard, L. & Despied, S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of escherichia coli: protective role of superoxide dismutase. Journal of bacteriology 177, 2305 (1995).

[41] Kohanski, M. A., DePristo, M. A. & Collins, J. J. Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Molecular Cell 37, 311–320 (2010).

[42] Nordstrom, D. Worldwide occurrences of arsenic in ground water. Science 296, 2143 (2002).

[43] Waters, C. & Bassler, B. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005).

[44] van der Meer, J. & Belkin, S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Reviews Microbiology 8, 511–522 (2010).

[45] Daunert, S. et al. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chemical Reviews 100, 2705–2738 (2000).

[46] Leveau, J. & Lindow, S. Bioreporters in microbial ecology. Current opinion in microbiology 5, 259–265 (2002).

[47] Mather, W., Bennett, M., Hasty, J. & Tsimring, L. Delay-Induced Degrade-and- Fire Oscillations in Small Genetic Circuits. Biophys. J Phys Rev Lett 102, 068105 (2009).

[48] Quan, J. & Tian, J. Circular polymerase extension cloning of complex gene libraries and pathways. PloS one 4, e6441 (2009).

[49] Stocker, J. et al. Development of a set of simple bacterial biosensors for quantitative and rapid measurements of arsenite and arsenate in potable water. Environ. Sci. Technol 37, 4743–4750 (2003).

[50] Keiler, K.,Waller, P. & Sauer, R. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger rna. Science 271, 990 (1996).