A multiplexed, electrochemical interface for gene-circuit-based sensors

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Abstract

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of functions in biological systems. To date, gene-circuit-based sensors have primarily used optical proteins (for example, fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene-circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors by demonstrating the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface for materials, hardware and software.

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Fig. 1: A gene-circuit/electrode interface for cell-free synthetic gene networks.
Fig. 2: Development of orthogonal, restriction-enzyme-based reporters.
Fig. 3: Electrochemical detection of restriction-enzyme reporters.
Fig. 4: Application of the gene-circuit/electrochemical interface for small-molecule- and RNA-actuated electrochemical signalling.
Fig. 5: Detection of mcr genes.

Data availability

All raw data presented in the manuscript are available upon request from the corresponding authors.

Code availability

All custom computer code used in the manuscript is available upon request from the corresponding authors.

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Acknowledgements

The molecular components of the ligand-inducible gene circuit were kindly provided by the Doktycz lab. The plasmid pSB3C5-proD-B0032-E0051 was a gift from J. Davis and R. Sauer (Addgene plasmid no. 107241). J.B.C. was funded by an Ontario Graduate Scholarship. This work was supported by the NSERC Discovery Grants Program (RGPIN-2016-06352), the CIHR Foundation Grant Program (201610FDN-375469), The University of Toronto’s Connaught New Research Award and the CIHR Canada Research Chair Program (950-231075) to K.P.; the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund (C1TPA-2016-06), and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI136571 (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health) to K.P. and S.O.K.; an NIH Director’s New Innovator Award (1DP2GM126892), an Arizona Biomedical Research Commission New Investigator Award (ADHS16-162400), an Alfred P. Sloan Research Fellowship (FG-2017-9108), Gates Foundation funds (OPP1160667), and Gordon and Betty Moore Foundation funds (no. 6984) to A.A.G. We thank S. Cicek for her help enhancing the high-throughput data analysis. We thank M. Labib and C. Nemr for their advice and support of the project.

Author information

P.S.M. designed and performed molecular experiments and co-wrote the manuscript; S.J.S. designed and performed electrochemical experiments and co-wrote the manuscript; J.B.C. designed and performed electrochemical experiments and co-wrote the manuscript; M.K. designed and performed molecular experiments and edited the manuscript; A.T. designed and performed molecular experiments and edited the manuscript; C.R. designed and performed molecular experiments; W.L. contributed to designing the DNA duplex reporter and electrochemical chip; D.M. designed the toehold switches; A.A.G. designed the toehold switches and edited the manuscript; S.O.K. was responsible for project supervision, designed experiments and edited the manuscript; K.P. was responsible for project design and supervision, designed experiments and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Correspondence to Shana O. Kelley or Keith Pardee.

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Supplementary Information

Supplementary Materials

Supplementary materials and methods, Figs. 1–18 and Tables 1–10.

Supplementary Sequence Information

DNA sequences used for this project.

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Sadat Mousavi, P., Smith, S.J., Chen, J.B. et al. A multiplexed, electrochemical interface for gene-circuit-based sensors. Nat. Chem. (2019) doi:10.1038/s41557-019-0366-y

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