Background Modularity is a crucial issue in the engineering world, as it enables engineers to achieve predictable outcomes when different components are interconnected. Synthetic Biology aims to apply key concepts of engineering to design and construct new biological systems that exhibit a predictable behaviour. Even if physical and measurement standards have been recently proposed to facilitate the assembly and characterization of biological components, real modularity is still a major research issue. The success of the bottom-up approach strictly depends on the clear definition of the limits in which biological functions can be predictable. Results The modularity of transcription-based biological components has been investigated in several conditions. First, the activity of a set of promoters was quantified in Escherichia coli via different measurement systems (i.e., different plasmids, reporter genes, ribosome binding sites) relative to an in vivo reference promoter. Second, promoter activity variation was measured when two independent gene expression cassettes were assembled in the same system. Third, the interchangeability of input modules (a set of constitutive promoters and two regulated promoters) connected to a fixed output device (a logic inverter) expressing GFP was evaluated. The three input modules provide tunable transcriptional signals that drive the output device. If modularity persists, identical transcriptional signals trigger identical GFP outputs. To verify this, all the input devices were individually characterized and then the input-output characteristic of the logic inverter was derived in the different configurations. Conclusions Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%). This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.
References
[1]
Andrianantoandro E, Basu S, Karig D, Weiss R (2006) Synthetic biology: new engineering rules for an emerging discipline. Molecular Systems Biology 2.
[2]
Endy D (2005) Foundations for engineering biology. Nature 438: 449–453.
[3]
Heinemann M, Panke S (2006) Synthetic biology - putting engineering into biology. Bioinformatics 22: 2790–2799.
[4]
Serrano L (2007) Synthetic biology: promises and challenges. Molecular Systems Biology 3.
[5]
Foundation TB. BioBricks standard biological parts. Accessed 2012 Jun 18.
[6]
Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, et al. (2010) BglBricks: a exible standard for biological part assembly. Journal of Biological Engineering 4.
[7]
French CE (2009) Synthetic biology and biomass conversion: a match made in heaven? Journal of the Royal Society Interface 6: S547–S558.
[8]
MIT. Registry of Standard Biological Parts. Accessed 2012 Jun 18.
[9]
Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26: 787–793.
[10]
Anderson JC, Clarke EJ, Arkin AP, Voigt CA (2006) Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355: 619–627.
[11]
Savage DF, Way J, Silver PA (2008) Defossiling fuel: how synthetic biology can transform biofuel production. ACS Chemical Biology 3: 13–16.
[12]
Kwok R (2010) Five hard truths for synthetic biology. Nature 463: 288–290.
[13]
Ellis T, Wang X, Collins JJ (2009) Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature Biotechnology 27: 465–471.
[14]
Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, et al. (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 3.
[15]
Kelly JR (2008) Tools and reference standards supporting the engineering and evolution of synthetic biological systems. Ph.D. thesis, Massachusetts Institute of Technology.
[16]
Martin L, Che A, Endy D (2009) Gemini, a bifunctional enzymatic and uorescent reporter of gene expression. PLoS ONE 4.
[17]
Zucca S, Pasotti L, Mazzini G, Cusella De Angelis MG, Magni P (2012) Characterization of an inducible promoter in different DNA copy number conditions. BMC Bioinformatics 13: S11.
[18]
International Genetically Engineered Machine (iGEM). Accessed 2012 Jun 18.
[19]
Sauro HM (2008) Modularity defined. Molecular Systems Biology 4.
[20]
Del Vecchio D, Ninfa AJ, Sontag ED (2008) Modular cell biology: retroactivity and insulation. Molecular Systems Biology 4.
[21]
Kobayashi H, Kaern M, Araki M, Chung K, Gardner TS, et al. (2004) Programmable cells: inter-facing natural and engineered gene networks. PNAS 101: 8414–8419.
[22]
Ro DK, Paradise EM, Ouellet M, Fisher K, Newman KL, et al. (2006) Production of the anti-malarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943.
[23]
Yokobayashi Y, Weiss R, Arnold F (2002) Directed evolution of a genetic circuit. PNAS 99: 16587–16591.
[24]
Anderson JC, Voigt CA, Arkin AP (2007) Environmental signal integration by a modular AND gate. Molecular Systems Biology 3.
[25]
Davis JH, Rubin AJ, Sauer RT (2011) Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research 39: 1131–1141.
[26]
Cox III RS, Dunlop MJ, Elowitz MB (2010) A synthetic three-color scaffold for monitoring genetic regulation and noise. Journal of Biological Engineering 4.
[27]
Hajimorad M, Gray PR, Keasling JD (2011) A framework and model system to investigate linear system behavior in Escherichia coli. Journal of Biological Engineering 5.
[28]
Wang B, Kitney RI, Joly N, Buck M (2011) Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2.
[29]
Elowitz MB, Leibler S (2000) A synthetic oscillatory network of transcriptional regulators. Nature 403: 335–338.
[30]
Pasotti L, Quattrocelli M, Galli D, Cusella De Angelis MG, Magni P (2011) Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology. Biotech-nology Journal 6: 784–795.
[31]
Kim KH, Sauro HM (2010) Fan-out in gene regulatory networks. Journal of Biological Engineering 4.
[32]
Salis HM, Mirsky E, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology 27: 946–950.
[33]
Pasotti L, Zucca S, Lupotto M, Cusella De Angelis MG, Magni P (2011) Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release. Journal of Biological Engineering 5.
[34]
Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, et al. (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of Biological Engineering 5.
[35]
Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25: 1203–1210.
[36]
Knight TF (2003) Idempotent vector design for standard assembly of biobricks. Technical re-port, MIT DSpace. Accessed 2012 Jun 18.
