Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits

Abstract

Multicellularity enables the growth of complex life forms as it allows for the specialization of cell types, differentiation and large-scale spatial organization. In a similar way, modular construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. To achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feedforward and feedback in vitro gene circuits, we then implement artificial signalling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.

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Fig. 1: Artificial multicellular structures.
Fig. 2: Gene induction by diffusing inducers.
Fig. 3: Pulse generation by an incoherent feedforward loop circuit.
Fig. 4: Stochastic differentiation of artificial cells.

Data availability

Raw data used for the generation of the figures are available from the authors upon request. Plasmids pSB1A3-AD009, pSB1A3-AD010 and pSB1A3-AD011 are available on Addgene.

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Acknowledgements

This work was supported by the European Research Council (grant agreement no. 694410, AEDNA) and the DFG Cluster of Excellence Nanosystems Initiative Munich (DFG EXC 4/3). A.D. acknowledges additional support by the DFG Research Training Group ‘Chemical Foundations of Synthetic Biology’ GRK 2062/1. The authors thank B. Tinao for her preliminary work on the artificial multicellular assemblies. They also thank D. Ziegler and J. List for their help with set-up construction, S. Sagredo and E. Falgenhauer for providing purified enzymes, and M. Schwarz-Schilling for helpful discussions. Correspondence and request for materials should be addressed to F.C.S.

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Contributions

A.D. and F.C.S. designed the experiments and wrote the manuscript. A.D. performed the experiments and analysed the data. A.D. and F.C.S. performed the modelling.

Corresponding author

Correspondence to Friedrich C. Simmel.

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Supplementary information

Supplementary Information

Supplementary methods, text, figures, tables, videos and references

Supplementary Video 1

This video shows the time lapse of Fig. 1b–c

Supplementary Video 2

Time lapse of the diffusion of DFHBI into a network of receivers containing a Spinach RNA transcription

Supplementary Video 3

This video shows 6 arrays containing the pulse propagation circuit, with different numbers of receivers

Supplementary Video 4

These videos show 2D networks containing the pulse generator

Supplementary Video 5

This video shows specifically a time-lapse of Fig. 3g

Supplementary Video 6

This video shows a differentiating network of one sender (indicated by S) and 4 receivers containing the circuit described in Fig. 4b, left panel

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Dupin, A., Simmel, F.C. Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits. Nature Chem 11, 32–39 (2019). https://doi.org/10.1038/s41557-018-0174-9

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