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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Cameron, D. E., Bashor, C. J. & Collins, J. J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).
Cheng, A. A. & Lu, T. K. Synthetic biology: an emerging engineering discipline. Annu. Rev. Biomed. Eng. 14, 155–178 (2012).
Fossati, E. et al. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 5, 3283 (2014).
Smanski, M. J. et al. Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135–149 (2016).
Green, A. A. et al. Complex cellular logic computation using ribocomputing devices. Nature 548, 117–121 (2017).
Yehl, K. & Lu, T. Scaling computation and memory in living cells. Curr. Opin. Biomed. Eng 4, 143–151 (2017).
Kitada, T., DiAndreth, B., Teague, B. & Weiss, R. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067 (2018).
Mao, N., Cubillos-Ruiz, A., Cameron, D. E. & Collins, J. J. Probiotic strains detect and suppress cholera in mice. Sci. Transl. Med. 10, eaao2586 (2018).
Kotula, J. W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl Acad. Sci. USA 111, 4838–4843 (2014).
Keasling, J. D. Synthetic biology and the development of tools for metabolic engineering. Metab. Eng. 14, 189–195 (2012).
Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J. & Swartz, J. R. An integrated cell-free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 4, 220–230 (2008).
Shin, J., Jardine, P. & Noireaux, V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth. Biol 1, 408–413 (2012).
Pardee, K. et al. Paper-based synthetic gene networks. Cell 159, 940–954 (2014).
Pardee, K. et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266 (2016).
Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259.e12 (2016).
Huang, A. et al. BioBitsTM explorer: a modular synthetic biology education kit. Sci. Adv. 4, eaat5105 (2018).
Stark, J. C. et al. BioBitsTM bright: a fluorescent synthetic biology education kit. Sci. Adv. 4, eaat5107 (2018).
Wen, K. Y. et al. A cell-free biosensor for detecting quorum sensing molecules in P. aeruginosa-infected respiratory samples. ACS Synth. Biol. 6, 2293–2301 (2017).
Takahashi, M. K. et al. A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nat. Commun. 9, 3347 (2018).
Alligrant, T. M., Nettleton, E. G. & Crooks, R. M. Lab on a chip electrochemical detection of individual DNA. Lab Chip 13, 349–354 (2013).
Fan, C., Plaxco, K. W. & Heeger, A. J. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl Acad. Sci. USA 100, 9134–9137 (2003).
Khan, H. U. et al. In situ, label-free DNA detection using organic transistor sensors. Adv. Mater. 22, 4452–4456 (2010).
Patolsky, F., Lichtenstein, A. & Willner, I. Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat. Biotechnol. 19, 253–257 (2001).
Slinker, J. D., Muren, N. B., Gorodetsky, A. A. & Barton, J. K. Multiplexed DNA-modified electrodes. J. Am. Chem. Soc. 132, 2769–2774 (2010).
Das, J. et al. An ultrasensitive universal detector based on neutralizer displacement. Nat. Chem. 4, 642–648 (2012).
Zuo, X., Xiao, Y. & Plaxco, K. W. High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices. J. Am. Chem. Soc. 131, 6944–6945 (2009).
Kuang, Z., Kim, S. N., Crookes-Goodson, W. J., Farmer, B. L. & Naik, R. R. Biomimetic chemosensor: designing peptide recognition elements for surface functionalization of carbon nanotube field effect transistors. ACS Nano 4, 452–458 (2010).
Liu, H., Xiang, Y., Lu, Y. & Crooks, R. M. Aptamer-based origami paper analytical device for electrochemical detection of adenosine. Angew. Chem. Int. Ed. 51, 6925–6928 (2012).
Das, J. & Kelley, S. O. Protein detection using arrayed microsensor chips: tuning sensor footprint to achieve ultrasensitive readout of CA-125 in serum and whole blood. Anal. Chem. 83, 1167–1172 (2011).
Tang, D., Yuan, R. & Chai, Y. Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer. Anal. Chem. 80, 1582–1588 (2008).
Sage, A. T., Besant, J. D., Lam, B., Sargent, E. H. & Kelley, S. O. Ultrasensitive electrochemical biomolecular detection using nanostructured microelectrodes. Acc. Chem. Res. 47, 2417–2425 (2014).
Li, J. J., Geyer, R. & Tan, W. Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. Nucleic Acids Res 28, e52 (2000).
Soleymani, L., Fang, Z., Sargent, E. H. & Kelley, S. O. Programming the detection limits of biosensors through controlled nanostructuring. Nat. Nanotechnol. 4, 844–848 (2009).
Karig, D. K., Iyer, S., Simpson, M. L. & Doktycz, M. J. Expression optimization and synthetic gene networks in cell-free systems. Nucleic Acids Res. 40, 3763–3774 (2012).
Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).
Zhang, J. et al. Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry. Sci. Rep. 8, 3705 (2018).
García, V. et al. Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006–2017). Int. J. Antimicrob. Agents 52, 104–108 (2018).
Zadeh, J. N. et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).
Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).
Davis, J. H., Rubin, A. J. & Sauer, R. T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).
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.
The authors declare no competing interests.
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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. 12, 48–55 (2020) doi:10.1038/s41557-019-0366-y