Design and analysis of a tunable synchronized oscillator

Journal of Biological Engineering

Floor Hugenholtz²  Mark WJ van Passel³  Vítor AP Martins dos Santos¹  Matthijn C Hesselman¹  Youri van Nuland¹  Ruben GA van Heck¹  Dorett I Odoni¹  Brendan M Ryback¹ 

[1] Systems and Synthetic Biology, Wageningen University, Wageningen, Netherlands;Netherlands Consortium for Systems Biology, University of Amsterdam, Amsterdam, Netherlands;Centre for Zoonoses and Environmental Microbiology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands

关键词: Delay differential equation;    Transcriptional feedback;    Genetic circuit;    Synchronized tunable oscillator;   

Others  :  805169
DOI  :  10.1186/1754-1611-7-26

 received in 2013-04-19, accepted in 2013-11-12,  发布年份 2013

【 摘 要 】

Background

The use of in silico simulations as a basis for designing artificial biological systems (and experiments to characterize them) is one of the tangible differences between Synthetic Biology and “classical” Genetic Engineering. To this end, synthetic biologists have adopted approaches originating from the traditionally non-biological fields of Nonlinear Dynamics and Systems & Control Theory. However, due to the complex molecular interactions affecting the emergent properties of biological systems, mechanistic descriptions of even the simplest genetic circuits (transcriptional feedback oscillators, bi-stable switches) produced by these methods tend to be either oversimplified, or numerically intractable. More comprehensive and realistic models can be approximated by constructing “toy” genetic circuits that provide the experimenter with some degree of control over the transcriptional dynamics, and allow for experimental set-ups that generate reliable data reflecting the intracellular biochemical state in real time. To this end, we designed two genetic circuits (basic and tunable) capable of exhibiting synchronized oscillatory green fluorescent protein (GFP) expression in small populations of Escherichia coli cells. The functionality of the basic circuit was verified microscopically. High-level visualizations of computational simulations were analyzed to determine whether the reliability and utility of a synchronized transcriptional oscillator could be enhanced by the introduction of chemically inducible repressors.

Results

Synchronized oscillations in GFP expression were repeatedly observed in chemically linked sub-populations of cells. Computational simulations predicted that the introduction of independently inducible repressors substantially broaden the range of conditions under which oscillations could occur, in addition to allowing the frequency of the oscillation to be tuned.

Conclusions

The genetic circuits described here may prove to be valuable research tools for the study of synchronized transcriptional feedback loops under a variety of conditions and experimental set-ups. We further demonstrate the benefit of using abstract visualizations to discover subtle non-linear trends in complex dynamic models with large parameter spaces.

【 授权许可】

   
2013 Ryback et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]De Jong H: Modeling and simulation of genetic regulatory systems: a literature review. J Comput Biol 2002, 9(1):67-103.
• [2]McAdams HH, Arkin A: Gene regulation: towards a circuit engineering discipline. Curr Biol 2000, 10(8):318-320.
• [3]Endy D: Foundations for engineering biology. Nature 2005, 438(7067):449-453.
• [4]Hasty J, McMillen D, Isaacs F, Collins JJ: Computational studies of gene regulatory networks: in numero molecular biology. Nat Rev Genet 2001, 2(4):268-279.
• [5]Krishnanathan K, Anderson SR, Billings SA, Kadirkamanathan V: A data-driven framework for identifying nonlinear dynamic models of genetic parts. ACS Synthetic Biol 2012, 1(8):375-384.
• [6]Chandran D, Sauro HM: Hierarchical model synthetic biol. ACS Synthetic Biol 2012, 1(8):353-364.
• [7]Gardner TS, Cantor CR, Collins JJ: Construction of a genetic toggle switch inEscherichia coli. Nature 2000, 403:339-342.
• [8]Atkinson MR, Savageau MA, Myers JT, Ninfa AJ: Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in escherichia coli. Cell 2003, 113(5):597-607.
• [9]Zhou T, Zhang J, Yuan Z, Chen L: Synchronization of genetic oscillators. Chaos: Interdiscipl J Nonlinear Sci 2008, 18(3):20.
• [10]Kim JR, Shin D, Jung SH, Heslop-Harrison P, Cho KH: A design principle underlying the synchronization of oscillations in cellular systems. J Cell Sci 2010, 123(4):537-543.
• [11]O’Brien EL, Van Itallie E, Bennett MR: Modeling synthetic gene oscillators. Math Biosci 2012, 236(1):1-15.
• [12]Purcell O, Savery NJ, Grierson CS, di Bernardo M: A comparative analysis of synthetic genetic oscillators. J Roy Soc Interface 2010, 7(52):1503-1524.
• [13]Elowitz MB, Leibler S: A synthetic oscillatory network of transcriptional regulators. Nature 2000, 403(6767):335-338.
• [14]Mondragón-Palomino O, Tsimring L, Hasty J: A synchronized quorum of genetic clocks. Nature 2010, 463(7279):326-330.
• [15]Nandagopal N, Elowitz MB: Synthetic biology: integrated gene circuits. Science 2011, 333(6047):1244-1248.
• [16]Sayut DJ, Sun L: Slow activator degradation reduces the robustness of a coupled feedback loop oscillator. Mol BioSystems 2010, 6(8):1469-1474.
• [17]Garcia-Ojalvo J, Elowitz MB, Strogatz SH: Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing. Proc Natl Acad Sci U S A 2004, 101(30):10955-10960.
• [18]Shetty RP, Endy D, Knight TF: Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2008, 2(1):1-12.
• [19]Hesselman MC, Odoni DI, Ryback BM, de Groot S, van Heck RGA, Keijsers J, Kolkman P, Nieuwenhuijse D, van Nuland YM, Sebus E, Spee R, de Vries H, Wapenaar MT, Ingham CJ, Schroën K, Santos VAP M d, Spaans SK, Hugenholtz F, van Passel MWJ: A multi-platform flow device for microbial (co-) cultivation and microscopic analysis. PloS One 2012, 7(5):e36982.
• [20]Ingham CJ, Sprenkels A, Bomer J, Molenaar D, van den Berg A, van Hylckama Vlieg JE, de Vos WM: The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms. Proc Nat Acad Sci 2007, 104(46):18217-18222.
• [21]Prindle A, Samayoa P, Razinkov I, Danino T, Tsimring LS, Hasty J: A sensing array of radically coupled genetic ‘biopixels’. Nature 2012, 481(7379):39-44.
• [22]Knight T: Idempotent vector design for standard assembly of biobricks. MIT Synthetic Biology Working Group 2003.