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Development of epistatic YES and AND protein logic gates and their assembly into signalling cascades

Nature Nanotechnology volume 18, pages 1327–1334 (2023)Cite this article

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Abstract

The construction and assembly of artificial allosteric protein switches into information and energy processing networks connected to both biological and non-biological systems is a central goal of synthetic biology and bionanotechnology. However, designing protein switches with the desired input, output and performance parameters is challenging. Here we use a range of reporter proteins to demonstrate that their chimeras with duplicated receptor domains produce YES gate protein switches with large (up to 9,000-fold) dynamic ranges and fast (minutes) response rates. In such switches, the epistatic interactions between largely independent synthetic allosteric sites result in an OFF state with minimal background noise. We used YES gate protein switches based on β-lactamase to develop quantitative biosensors of therapeutic drugs and protein biomarkers. Furthermore, we demonstrated the reconfiguration of YES gate switches into AND gate switches controlled by two different inputs, and their assembly into signalling networks regulated at multiple nodes.

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Fig. 1: YES gate allosteric switch based on TEM-1 BLA.
Fig. 2: Biosensors based on CaM-BLA YES gate switch.
Fig. 3: Construction of allosteric CaM-TreA chimeras.
Fig. 4: Construction of YES-gate-based and AND-gate-based signalling cascades.

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Data availability

The authors declare that the data supporting the findings of this study are available in this paper and its supplementary information files. The following PDB entries were used in this study: BLA inhibitory protein I in complex with TEM-1 BLA (3gmw), trehalase (2jg0) and TEM-1 BLA in complex with inhibitor (1ERQ). Source data are provided with this paper.

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Acknowledgements

We thank C. Pretorius from Pathology Queensland for carrying out the quantification of biomarkers in human samples for this study. We thank D. Massana Roquero from Stanford University School of Medicine for help with experiments related to alginate gel. We also thank I. Berezovsky from A-STAR Singapore and S. Fleishman from Weizmann Institute for stimulating discussions. This work was supported in part by the Australian Research Council Discovery Projects (DP160100973 and DP150100936), as well as ARC Centre of Excellence in Synthetic Biology (CE200100029 to K.A.). The work was also supported by National Health and Medical Research Council Development Grants (APP1113262 to K.A. and APP1179001 to K.A. and J.P.J.U.). This work was also in part supported by the Human Frontier Science Program (grant RGP0002/2018 to O.S., K.A. and E.K.) and the US Department of Defense (grant W81XWH-20-1-0708 to K.A. and E.K.). K.A. gratefully acknowledges the financial support of the Synthetic Biology Alliance between the Commonwealth Scientific and Industrial Research Organisation (CSIRO) and Queensland University of Technology.

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Authors and Affiliations

  1. ARC Centre of Excellence in Synthetic Biology, Brisbane, Queensland, Australia

    Zhong Guo, Claudia E. Vickers & Kirill Alexandrov

  2. Centre for Agriculture and the Bioeconomy, Queensland University of Technology, Brisbane, Queensland, Australia

    Zhong Guo, Cagla Ergun Ayva, Patricia Walden, Claudia E. Vickers & Kirill Alexandrov

  3. School of Biology and Environmental Science, Queensland University of Technology, Brisbane, Queensland, Australia

    Zhong Guo, Cagla Ergun Ayva, Patricia Walden, Claudia E. Vickers & Kirill Alexandrov

  4. Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY, USA

    Oleh Smutok & Evgeny Katz

  5. Yakka Bio, Canberra, New South Wales, Australia

    Jake Parker

  6. UNSW Founders, University of New South Wales, Sydney, New South Wales, Australia

    Jason Whitfield

  7. Department of Chemical Pathology, Pathology Queensland, Brisbane, Queensland, Australia

    Jacobus P. J. Ungerer

  8. Faculty of Health and Behavioural Sciences, University of Queensland, Brisbane, Queensland, Australia

    Jacobus P. J. Ungerer

  9. CSIRO-QUT Synthetic Biology Alliance, Brisbane, Queensland, Australia

    Kirill Alexandrov

  10. Centre for Genomics and Personalised Health, Queensland University of Technology, Brisbane, Queensland, Australia

    Kirill Alexandrov

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  1. Zhong Guo
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Contributions

Z.G. designed experiments, performed experiments, analysed data and contributed to writing the paper. O.S. designed experiments, performed experiments, analysed data and contributed to writing the paper. C.E.A. designed experiments, performed experiments, analysed data and contributed to writing the paper. P.W. performed experiments. J.P. designed experiments, analysed data and contributed to writing the paper. J.W. designed experiments, performed experiments, analysed data and contributed to writing the paper. C.E.V. designed experiments, performed experiments, analysed data and contributed to writing the paper. J.P.J.U. designed experiments and analysed data. E.K. designed experiments, analysed data and contributed to writing the paper. K.A. designed experiments, analysed data and contributed to writing the paper.

Corresponding author

Correspondence to Kirill Alexandrov.

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Competing interests

The authors declare the following competing interests: Z.G. and K.A. are named inventors on PCT patent application PCT/IB2023/020001 entitled ‘AND-gate protein-based switches’ filed by Queensland University of Technology that covers the YES biosensor architecture used in this study. The other authors declare no competing interests.

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Nature Nanotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–6.

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Supplementary Data 1

Data used to generate plots and graphs in Supplementary Figs. 1–10. Unprocessed SDS–PAGE gels used to generate Fig. 3i.

Source data

Source Data Figs. 1–4

Data used to generate plots and graphs in Figs. 1–4.

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Guo, Z., Smutok, O., Ayva, C.E. et al. Development of epistatic YES and AND protein logic gates and their assembly into signalling cascades. Nat. Nanotechnol. 18, 1327–1334 (2023). https://doi.org/10.1038/s41565-023-01450-y

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