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A Simple Protein Synthesis Model for the PURE System Operation

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Abstract

The encapsulation of transcription–translation (TX–TL) cell-free machinery inside lipid vesicles (liposomes) is a key element in synthetic cell technology. The PURE system is a TX–TL kit composed of well-characterized parts, whose concentrations are fine tunable, which works according to a modular architecture. For these reasons, the PURE system perfectly fulfils the requirements of synthetic biology and is widely used for constructing synthetic cells. In this work, we present a simplified mathematical model to simulate the PURE system operations. Based on Michaelis–Menten kinetics and differential equations, the model describes protein synthesis dynamics by using 9 chemical species, 6 reactions and 16 kinetic parameters. The model correctly predicts the time course for messenger RNA and protein production and allows quantitative predictions. By means of this model, it is possible to foresee how the PURE system species affect the mechanism of proteins synthesis and therefore help in understanding scenarios where the concentration of the PURE system components has been modified purposely or as a result of stochastic fluctuations (for example after random encapsulation inside vesicles). The model also makes the determination of response coefficients for all species involved in the TX–TL mechanism possible and allows for scrutiny on how chemical energy is consumed by the three PURE system modules (transcription, translation and aminoacylation).

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References

  • Atkinson DE (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7(11):4030

    Article  Google Scholar 

  • Calviello L, Stano P, Mavelli F, Luisi PL, Marangoni R (2013) Quasi-cellular systems: stochastic simulation analysis at nanoscale range. BMC Bioinform 14(Suppl 7):S7

    Google Scholar 

  • Chiarabelli C, Stano P, Luisi PL (2009) Chemical approaches to synthetic biology. Curr Opin Biotechnol 20(4):492

    Article  Google Scholar 

  • Choudhury A, Hodgman CE, Anderson MJ, Jewett MC (2014) Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis. Biochem Eng J 91:140

    Article  Google Scholar 

  • Dominak LM, Keating CD (2007) Polymer encapsulation within giant lipid vesicles. Langmuir 23(13):7148

    Article  Google Scholar 

  • Dong H, Nilsson L, Kurland CG (1996) Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J Mol Biol 260(5):649

    Article  Google Scholar 

  • Endy D (2005) Foundations for engineering biology. Nature 438(7067):449

    Article  Google Scholar 

  • Frazier JM, Chushak Y, Foy B (2009) Stochastic simulation and analysis of biomolecular reaction networks. BMC Syst Biol 3:64

    Article  Google Scholar 

  • Harris DC, Jewett MC (2012) Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry. Curr Opin Biotechnol 23(5):672

    Article  Google Scholar 

  • Heinonen JK (2001) Biological role of inorganic pyrophosphate. Kluver Academic Publisher, New York

    Book  Google Scholar 

  • Hockenberry AJ, Jewett MC (2012) Synthetic in vitro circuits. Curr Opin Chem Biol 16(3–4):253

    Article  Google Scholar 

  • Hodgman CE, Jewett MC (2012) Cell-free synthetic biology: thinking outside the cell. Metab Eng 14(3):261

    Article  Google Scholar 

  • Jewett MC, Fritz BR, Timmerman LE, Church GM (2013) In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation. Mol Syst Biol 9(1):678

    Article  Google Scholar 

  • Karzbrun E, Shin J, Bar-Ziv RH, Noireaux V (2011) Coarse-grained dynamics of protein synthesis in a cell-free system. Phys Rev Lett 106(4):048104

    Article  Google Scholar 

  • Kato A, Yanagisawa M, Sato YT, Fujiwara K, Yoshikawa K (2012) Cell-sized confinement in microspheres accelerates the reaction of gene expression. Sci Rep 2:283

    Google Scholar 

  • Kazuta Y, Matsuura T, Ichihashi N, Yomo T (2014) Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system. J Biosci Bioeng 118(5):554

    Article  Google Scholar 

  • Kuruma Y, Stano P, Ueda T, Luisi PL (2009) A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim Biophys Acta 1788(2):567

    Article  Google Scholar 

  • Lazzerini-Ospri L, Stano P, Luisi P, Marangoni R (2012) Characterization of the emergent properties of a synthetic quasi-cellular system. BMC Bioinform 13(Suppl 4):S9

    Article  Google Scholar 

  • LeDuc PR, Wong MS, Ferreira PM, Groff RE, Haslinger K, Koonce MP, Lee WY, Love JC, McCammon JA, Monteiro-Riviere NA, Rotello VM, Rubloff GW, Westervelt R, Yoda M (2007) Towards an in vivo biologically inspired nanofactory. Nat Nano 2(1):3

    Article  Google Scholar 

  • Liu AP, Fletcher DA (2009) Biology under construction: in vitro reconstitution of cellular function. Nat Rev Mol Cell Biol 10(9):644

    Article  Google Scholar 

  • Lohse B, Bolinger PY, Stamou D (2008) Encapsulation efficiency measured on single small unilamellar vesicles. J Am Chem Soc 130(44):14372

    Article  Google Scholar 

  • Luisi PL, Ferri F, Stano P (2006) Approaches to semi-synthetic minimal cells: a review. Naturwissenschaften 93(1):1

    Article  Google Scholar 

  • Luisi PL, Allegretti M, Pereira de Souza T, Steiniger F, Fahr A, Stano P (2010) Spontaneous protein crowding in liposomes: a new vista for the origin of cellular metabolism. ChemBioChem 11(14):1989

    Article  Google Scholar 

  • Matsubayashi H, Ueda T (2014) Purified cell-free systems as standard parts for synthetic biology. Curr Opin Chem Biol 22:158

    Article  Google Scholar 

  • Matsubayashi H, Kuruma Y, Ueda T (2014) In vitro synthesis of the E. coli sec translocon from DNA. Angew Chem Int Ed 53(29):7535

    Article  Google Scholar 

  • Matsuura T, Kazuta Y, Aita T, Adachi J, Yomo T (2009) Quantifying epistatic interactions among the components constituting the protein translation system. Mol Syst Biol 5:297

    Article  Google Scholar 

  • Matsuura T, Hosoda K, Kazuta Y, Ichihashi N, Suzuki H, Yomo T (2012) Effects of compartment size on the kinetics of intracompartmental multimeric protein synthesis. ACS Synth Biol 1(9):431

    Article  Google Scholar 

  • Matveev SV, Vinokurov LM, Shaloiko LA, Davies C, Matveeva EA, Alakhov YuB (1996) Effect of the ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system. Biochim Et Biophys Acta 1293(2):207

    Article  Google Scholar 

  • Mavelli F, Altamura E, Cassidei L, Stano P (2014) Recent theoretical approaches to minimal artificial cells. Entropy 16(5):2488–2511

    Article  Google Scholar 

  • Murtas G, Kuruma Y, Bianchini P, Diaspro A, Luisi PL (2007) Protein synthesis in liposomes with a minimal set of enzymes. Biochem Biophys Res Commun 363(1):12

    Article  Google Scholar 

  • NakanoT Moore M, Enomoto A, Suda T (2011) Biological functions for information and communication technologies. In: Sawai H (ed) Studies in computational intelligence, no. 320. Springer, Berlin, pp 49–86

    Google Scholar 

  • Nishimura K, Matsuura T, Nishimura K, Sunami T, Suzuki H, Yomo T (2012) Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry. Langmuir 28(22):8426

    Article  Google Scholar 

  • Niwa T, Kanamori T, Ueda T, Taguchi H (2012) Global analysis of chaperone effects using a reconstituted cell-free translation system. Proc Nat Acad Sci 109(23):8937

    Article  Google Scholar 

  • Noireaux V, Bar-Ziv R, Libchaber A (2003) Principles of cell-free genetic circuit assembly. Proc Natl Acad Sci USA 100(22):12672

    Article  Google Scholar 

  • Okano T, Matsuura T, Suzuki H, Yomo T (2014) Cell-free protein synthesis in a microchamber revealed the presence of an optimum compartment volume for high-order reactions. ACS Synth Biol 3(6):347

    Article  Google Scholar 

  • Pereira de Souza T, Stano P, Luisi PL (2009) The minimal size of liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis. ChemBioChem 10(6):1056

    Article  Google Scholar 

  • Pereira de Souza T, Steiniger F, Stano P, Fahr A, Luisi PL (2011) Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism. ChemBioChem 12(15):2325

    Article  Google Scholar 

  • Rampioni G, Mavelli F, Damiano L, DAngelo F, Messina M, Leoni L, Stano P (2014) A synthetic biology approach to bio-chem-ICT: first moves towards chemical communication between synthetic and natural cells. Nat Comput 13:333–349

  • Saito H, Kato Y, Le Berre M, Yamada A, Inoue T, Yosikawa K, Baigl D (2009) Time-resolved tracking of a minimum gene expression system reconstituted in giant liposomes. ChemBioChem 10(10):1640

    Article  Google Scholar 

  • Schoborg JA, Hodgman CE, Anderson MJ, Jewett MC (2014) Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis. Biotechnol J 9(5):630

    Article  Google Scholar 

  • Seo SW, Yang J, Min BE, Jang S, Lim JH, Lim HG, Kim SC, Kim SY, Jeong JH, Jung GY (2013) Synthetic biology: tools to design microbes for the production of chemicals and fuels. Biotechnol Adv 31(6):811

    Article  Google Scholar 

  • Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19(8):751. doi:10.1038/90802

    Article  Google Scholar 

  • Shimizu Y, Kanamori T, Ueda T (2005) Protein synthesis by pure translation systems. Methods 36(3):299

    Article  Google Scholar 

  • Shin J, Jardine P, Noireaux V (2012) Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol 1(9):408

    Article  Google Scholar 

  • Siegal-Gaskins D, Tuza ZA, Kim J, Noireaux V, Murray RM (2014) Gene circuit performance characterization and resource usage in a cell-free “breadboard”. ACS Synth Biol 3(6):416

    Article  Google Scholar 

  • Soga H, Fujii S, Yomo T, Kato Y, Watanabe H, Matsuura T (2014) In vitro membrane protein synthesis inside cell-sized vesicles reveals the dependence of membrane protein integration on vesicle volume. ACS Synth Biol 3:372–379

    Article  Google Scholar 

  • Stano P, Carrara P, Kuruma Y, de Souza TP, Luisi PL (2011) Compartmentalized reactions as a case of soft-matter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles. J Mater Chem 21(47):18887

    Article  Google Scholar 

  • Stano P, Rampioni G, Carrara P, Damiano L, Leoni L, Luisi PL (2012) Semi-synthetic minimal cells as a tool for biochemical ICT. BioSystems 109(1):24

    Article  Google Scholar 

  • Stano P, Luisi PL (2013) Semi-synthetic minimal cells: origin and recent developments. Curr Opin Biotechnol 24:633–638

    Article  Google Scholar 

  • Stano P, D’Aguanno E, Bolz J, Fahr A, Luisi PL (2013) A remarkable self-organization process as the origin of primitive functional cells. Angew Chem Int Ed Engl 52(50):13397

    Article  Google Scholar 

  • Stano P, Souza T, Carrara P, Altamura E, D’Aguanno E, Caputo M, Luisi PL, Mavelli F (2015) Recent biophysical issues about the preparation of solute-filled lipid vesicles. Mech Adv Mater Struct 22:748–759

    Article  Google Scholar 

  • Stögbauer T, Windhager L, Zimmer R, Rädler JO (2012) Experiment and mathematical modeling of gene expression dynamics in a cell-free system. Integr Biol 4(5):494

    Article  Google Scholar 

  • Sun BY, Chiu DT (2005) Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection. Anal Chem 77(9):2770

    Article  Google Scholar 

  • Sunami T, Hosoda K, Suzuki H, Matsuura T, Yomo T (2010) Cellular compartment model for exploring the effect of the lipidic membrane on the kinetics of encapsulated biochemical reactions. Langmuir 26(11):8544

    Article  Google Scholar 

  • van Nies P, Nourian Z, Kok M, van Wijk R, Moeskops J, Westerlaken I, Poolman JM, Eelkema R, van Esch JH, Kuruma Y, Ueda T, Danelon C (1963) Unbiased tracking of the progression of mRNA and protein synthesis in bulk and in liposome-confined reactions. ChemBioChem 14(15):1963–1966

    Article  Google Scholar 

  • Walde P, Umakoshi H, Stano P, Mavelli F (2014) Emergent properties arising from the assembly of amphiphiles. Artificial vesicle membranes as reaction promoters and regulators. Chem Comm 50:10177–10197

    Article  Google Scholar 

  • Yarchuk O, Jacques N, Guillerez J, Dreyfus M (1992) Interdependence of translation, transcription and mRNA degradation in the lacZ gene. J Mol Biol 226(3):581

    Article  Google Scholar 

  • Zhao H (ed) (2013) Synthetic Biology. Tools and applications. Academic Press-Elsevier, Amsterdam

Download references

Acknowledgments

We are grateful to Pier Luigi Luisi for his guidance in the field of synthetic cells and for useful comments on the manuscript. The modeling work has been started within the PRIN2008 (2008FY7RJ4) Synthetic Cells project and further expanded thanks to networking initiatives such EU-COST Actions CM0703 (Systems Chemistry) and CM1304 (Emergence and Evolution of Complex Chemical Systems). We thank Margherita Caputo (Univ. Bari) and Francesca D’Angelo (Roma Tre Univ.) for their involvement in the initial phase of the work.

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Correspondence to Pasquale Stano.

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Mavelli, F., Marangoni, R. & Stano, P. A Simple Protein Synthesis Model for the PURE System Operation. Bull Math Biol 77, 1185–1212 (2015). https://doi.org/10.1007/s11538-015-0082-8

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