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Building in vitro transcriptional regulatory networks by successively integrating multiple functional circuit modules

Nature Chemistry volume 11, pages 829–838 (2019)Cite this article

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Abstract

The regulation of cellular dynamics and responses to stimuli by genetic regulatory networks suggests how in vitro chemical reaction networks might analogously direct the dynamics of synthetic materials or chemistries. A key step in developing genetic regulatory network analogues capable of this type of sophisticated regulation is the integration of multiple coordinated functions within a single network. Here, we demonstrate how such functional integration can be achieved using in vitro transcriptional genelet circuits that emulate essential features of cellular genetic regulatory networks. By successively incorporating functional genelet modules into a bistable circuit, we construct an integrated regulatory network that dynamically changes its state in response to upstream stimuli and coordinates the timing of downstream signal expression. We use quantitative models to guide module integration and develop strategies to mitigate undesired interactions between network components that arise as the size of the network increases. This approach could enable the construction of in vitro networks capable of multifaceted chemical and material regulation.

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Fig. 1: Genelets and a switchable bistable network.
Fig. 2: Inducer RNAs switch the state of the bistable network.
Fig. 3: Upstream nodes that produce inducer RNAs switch the state of the iBN.
Fig. 4: Connecting the iBN-uA to downstream nodes enables switching between the production of different downstream RNA signals.
Fig. 5: A fan-out feed-forward architecture enables mutually exclusive downstream signal expression states.

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Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request.

Code availability

The MATLAB code that was used in the Supplementary Information to conduct the simulations presented in this study is available from the corresponding authors on reasonable request.

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Acknowledgements

The authors would like to thank E. Franco, L. Green, H. Subramanian and C. C. Samaniego for their help with the initial genelet experiments, and J. Fern for insightful conversations. S.S. was supported by National Science Foundation Graduate Research Fellowship DGE-1746891. Materials, supplies and R.S. were supported by the Department of Energy BES DE-SC001 0426.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Samuel W. Schaffter & Rebecca Schulman

  2. Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA

    Rebecca Schulman

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S.W.S. conceived and designed the study, designed and performed the experiments, analysed and interpreted the data, and wrote the manuscript. R.S. conceived and supervised the study, interpreted the data and wrote the manuscript.

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Correspondence to Samuel W. Schaffter or Rebecca Schulman.

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Schaffter, S.W., Schulman, R. Building in vitro transcriptional regulatory networks by successively integrating multiple functional circuit modules. Nat. Chem. 11, 829–838 (2019). https://doi.org/10.1038/s41557-019-0292-z

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