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Synthetic circuits integrating logic and memory in living cells

Abstract

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

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Figure 1: Integrated logic and memory devices.
Figure 2: Recombinase-based logic gates can implement a complete set of two-input–one-output Boolean logic gates without needing to cascade multiple universal gates together.
Figure 3: Stable memory maintenance over multiple cell generations and after cell death.
Figure 4: Recombinase-based logic and memory can implement digital-to-analog converters.

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Acknowledgements

The bxb1 gene was a generous gift from G.F. Hatfull (Department of Biological Sciences, University of Pittsburgh), and the riboregulator plasmids were donated by J.J. Collins (Biomedical Engineering, Boston University). The authors thank R. Danial and A.A. Cheng for careful comments on the manuscript. This work was supported by an Office of Naval Research Multidisciplinary University Research Initiative (MURI) grant and the Defense Advanced Research Projects Agency (DARPA).

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T.K.L. conceived of this study. P.S. and J.Y. implemented, constructed and performed all experiments. All authors analyzed the data, discussed results and wrote the manuscript.

Corresponding author

Correspondence to Timothy K Lu.

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Competing interests

P.S., J.Y. and T.K.L. have filed a provisional application with the US Patent and Trademark Office on this work.

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Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Data (PDF 6618 kb)

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Siuti, P., Yazbek, J. & Lu, T. Synthetic circuits integrating logic and memory in living cells. Nat Biotechnol 31, 448–452 (2013). https://doi.org/10.1038/nbt.2510

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