Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.
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Carlson, E. D., Gan, R., Hodgman, C. E. & Jewett, M. C. Cell-free protein synthesis: applications come of age. Biotechnol. Adv. 30, 1185–1194 (2012).
Smith, M. T., Wilding, K. M., Hunt, J. M., Bennett, A. M. & Bundy, B. C. The emerging age of cell-free synthetic biology. FEBS Lett. 588, 2755–2761 (2014).
Hodgman, C. E. & Jewett, M. C. Cell-free synthetic biology: thinking outside the cell. Metab. Eng. 14, 261–269 (2012).
Miller, D. & Gulbis, J. Engineering protocells: prospects for self-assembly and nanoscale production-lines. Life 5, 1019–1053 (2015).
Shimizu, Y., Kuruma, Y., Ying, B.-W., Umekage, S. & Ueda, T. Cell-free translation systems for protein engineering. FEBS J. 273, 4133–4140 (2006).
Shin, J. & Noireaux, V. An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. ACS Synth. Biol. 1, 29–41 (2012).
Takahashi, M. K. et al. Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription–translation (TX-TL) systems. ACS Synth. Biol. 4, 503–515 (2015).
Michener, J. K., Thodey, K., Liang, J. C. & Smolke, C. D. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. Metab. Eng. 14, 212–222 (2012).
Vamvakaki, V. & Chaniotakis, N. A. Pesticide detection with a liposome-based nano-biosensor. Biosens. Bioelectron. 22, 2848–2853 (2007).
Pardee, K. et al. Paper-based synthetic gene networks. Cell 159, 940–954 (2014).
Lentini, R. et al. Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour. Nat. Commun. 5, 4012 (2014).
Zemella, A., Thoring, L., Hoffmeister, C. & Kubick, S. Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. Chem. Bio. Chem. 16, 2420–31 (2015).
Forster, A. C. & Church, G. M. Towards synthesis of a minimal cell. Mol. Syst. Biol. 2, 45 (2006).
Brea, R. J., Hardy, M. D. & Devaraj, N. K. Towards self-assembled hybrid artificial cells: novel bottom-up approaches to functional synthetic membranes. Chem. Pub. Soc. Euro. 21, 12564–12570 (2015).
Luisi, P. L., Ferri, F. & Stano, P. Approaches to semi-synthetic minimal cells: a review. Naturwissenschaften 93, 1–13 (2006).
Stano, P. & Luisi, P. L. Semi-synthetic minimal cells: origin and recent developments. Curr. Opin. Biotechnol. 24, 633–638 (2013).
Murtas, G., Kuruma, Y., Bianchini, P., Diaspro, A. & Luisi, P. L. Protein synthesis in liposomes with a minimal set of enzymes. Biochem. Biophys. Res. Commun. 363, 12–17 (2007).
Yu, W. et al. Synthesis of functional protein in liposome. J. Biosci. Bioeng. 92, 590–593 (2001).
Oberholzer, T., Nierhaus, K. H. & Luisi, P. L. Protein expression in liposomes. Biochem. Biophys. Res. Commun. 261, 238–241 (1999).
Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl Acad. Sci. USA 101, 17669–17674 (2004).
Stech, M. et al. Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system. J. Biotechnol. 164, 220–231 (2012).
Weber, L. A, Feman, E. R. & Baglioni, C. A cell free system from HeLa cells active in initiation of protein synthesis. Biochemistry 14, 5315–5321 (1975).
Wimmer, E. Cell-free, de novo synthesis of poliovirus. Science. 254, 1647–1651, (1991).
Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S. & Imataka, H. An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46, 348–357 (2006).
Mikami, S., Kobayashi, T., Masutani, M., Yokoyama, S. & Imataka, H. A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr. Purif. 62, 190–198 (2008).
Tan, C., Saurabh, S., Bruchez, M. P., Schwartz, R. & Leduc, P. Molecular crowding shapes gene expression in synthetic cellular nanosystems. Nat. Nanotechnol. 8, 602–608 (2013).
de Souza, T. P. et al. Encapsulation of ferritin, ribosomes, and ribo-peptidic complexes inside liposomes: insights into the origin of metabolism. Orig. Life Evol. Biosph. 42, 421–428 (2012).
de Souza, T. P., Fahr, A., Luisi, P. L. & Stano, P. Spontaneous encapsulation and concentration of biological macromolecules in liposomes: an intriguing phenomenon and its relevance in origins of life. J. Mol. Evol. 79, 179–192 (2014).
Caschera, F. & Noireaux, V. Integration of biological parts toward the synthesis of a minimal cell. Curr. Opin. Chem. Biol. 22, 85–91 (2014).
Stefureac, R., Long, Y. T., Kraatz, H. B., Howard, P. & Lee, J. S. Transport of α-helical peptides through α-hemolysin and aerolysin pores. Biochemistry 45, 9172–9179 (2006).
Gouaux, E., Hobaugh, M. & Song, L. α-Hemolysin, γ-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure. Protein Sci. 6, 2631–2635 (1997).
Selgrade, D. F., Lohmueller, J. J., Lienert, F. & Silver, P. A. Protein scaffold-activated protein trans-splicing in mammalian cells. 135, 7713–7719 (2013).
Tu, Y. et al. Mimicking the cell: bio-inspired functions of supramolecular assemblies. Chem. Rev. 116, 2023–2078 (2016).
Del Vecchio, D., Ninfa, A. J. & Sontag, E. D. Modular cell biology: retroactivity and insulation. Mol. Syst. Biol. 4, 161 (2008).
Caschera, F. et al. Programmed vesicle fusion triggers gene expression. Langmuir 27, 13082–13090 (2011).
Meyenberg, K., Lygina, A. S., van den Bogaart, G., Jahn, R. & Diederichsen, U. SNARE derived peptide mimic inducing membrane fusion. Chem. Commun. 47, 9405–9407 (2011).
Robson Marsden, H., Korobko, A. V., Zheng, T., Voskuhl, J. & Kros, A. Controlled liposome fusion mediated by SNARE protein mimics. Biomater. Sci. 1, 1046–1054 (2013).
Inglés-Prieto, Á. et al. Light-assisted small-molecule screening against protein kinases. Nat. Chem. Biol. 11, 952–954 (2015).
Boyden, E. S. A history of optogenetics: the development of tools for controlling brain circuits with light. F1000 Biol. Rep. 3, 11 (2011).
Hanczyc, M. M., Fujikawa, S. M. & Szostak, J. W. Experimental models of primitive cellular compartments: encapsulation, growth, and division. Science 302, 618–622 (2003).
Adamala, K. & Szostak, J. W. Competition between model protocells driven by an encapsulated catalyst. Nat. Chem. 5, 495–501 (2013).
Balaram, P. Synthesizing life. Curr. Sci. 85, 1509–1510 (2003).
Adamala, K. et al. Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach. Comput. Struct. Biotechnol. J. 9, e201402004 (2014).
Ruiz-Mirazo, K., Briones, C. & de la Escosura, A. Prebiotic systems chemistry: new perspectives for the origins of life. Chem. Rev. 114, 285–366 (2014).
Glansdorff, N., Xu, Y. & Labedan, B. The last universal common ancestor: emergence, constitution and genetic legacy of an elusive forerunner. Biol. Direct 3, 29 (2008).
Woese, C. The universal ancestor. Proc. Natl Acad. Sci. USA 95, 6854–6859 (1998).
Theobald, D. L. A formal test of the theory of universal common ancestry. Nature 465, 219–222 (2010).
Spencer, A. C., Torre, P. & Mansy, S. S. The encapsulation of cell-free transcription and translation machinery in vesicles for the construction of cellular mimics. J. Vis. Exp. 80, e51304 (2013).
Adamala, K., Engelhart, A. E., Kamat, N. P., Jin, L. & Szostak, J. W. Construction of a liposome dialyzer for the preparation of high-value, small-volume liposome formulations. Nat. Protoc. 10, 927–938 (2015).
Shin, J. & Noireaux, V. Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70. J. Biol. Eng. 4, 8 (2010).
Lutz, R. & Bujard, H. 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 Res. 25, 1203–1210 (1997).
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).
Sun, Z. Z. et al. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. J. Vis. Exp. 79, e50762 (2013).
We thank E. Vasile and F. Chen for help with the SIM microscopy, and G. Paradis and K. Piatkevich for help with the flow-cytometry experiments. We thank N. Kamat and L. Jin for help with troubleshooting the DLS machine. We thank J. Szostak for sharing the liposome encapsulation formula. We thank V. Noireaux, A. Mershin and A. Engelhart for helpful discussions about cell-free TX/TL systems. E.S.B. acknowledges, for funding, the National Institutes of Health (NIH) 1U01MH106011, Jeremy and Joyce Wertheimer, NIH 1RM1HG008525, the Picower Institute Innovation Fund, NIH 1R01MH103910, NIH 1R01NS075421, National Science Foundation CBET 1053233, New York Stem Cell Foundation-Robertson Award and NIH Director's Pioneer Award 1DP1NS087724. D.A.M.-A. acknowledges support from the Janet and Sheldon Razin (1959) Fellowship.
K.P.A., D.A.M.-A. and E.S.B. submitted a provisional patent application based on this work.
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Adamala, K., Martin-Alarcon, D., Guthrie-Honea, K. et al. Engineering genetic circuit interactions within and between synthetic minimal cells. Nature Chem 9, 431–439 (2017). https://doi.org/10.1038/nchem.2644
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