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Engineering genetic circuit interactions within and between synthetic minimal cells

Abstract

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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Figure 1: An overview of genetic circuit interactions within and between synells.
Figure 2: Molecular confinement of multicomponent genetic cascades.
Figure 3: Comparison of single- and multicomponent genetic circuits.
Figure 4: Insulation of genetic circuits that operate in parallel liposome populations.
Figure 5: Communication between genetic circuits that operate in multiple liposome populations.
Figure 6: Fusion of complementary genetic circuits.

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Acknowledgements

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.

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K.P.A. and D.A.M.-A. contributed equally to this work. K.P.A., D.A.M.-A. and K.R.G.-H. performed the experiments. K.P.A., D.A.M.-A. and E.S.B. designed experiments, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Edward S. Boyden.

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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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