A sensing array of radically coupled genetic ‘biopixels’

Abstract

Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony ‘biopixels’ over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field.

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Figure 1: Sensing array of radically coupled genetic biopixels.
Figure 2: Frequency-modulated genetic biosensor.
Figure 3: Computational modelling of radical synchronization and biosensing.
Figure 4: Radical synchronization on a macroscopic scale.

References

  1. 1

    Gibson, D. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Hasty, J., McMillen, D. & Collins, J. J. Engineered gene circuits. Nature 420, 224–230 (2002)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Sprinzak, D. & Elowitz, M. B. Reconstruction of genetic circuits. Nature 438, 443–448 (2005)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Endy, D. Foundations for engineering biology. Nature 438, 449–453 (2005)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Ellis, T., Wang, X. & Collins, J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature Biotechnol. 27, 465–471 (2009)

    CAS  Article  Google Scholar 

  6. 6

    Kobayashi, H. et al. Programmable cells: interfacing natural and engineered gene networks. Proc. Natl Acad. Sci. USA 101, 8414–8419 (2004)

    ADS  CAS  Article  Google Scholar 

  7. 7

    You, L., Cox, R. S., III, Weiss, R. & Arnold, F. H. Programmed population control by cell–cell communication and regulated killing. Nature 428, 868–871 (2004)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–1134 (2005)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Mukherji, S. & Van Oudenaarden, A. Synthetic biology: understanding biological design from synthetic circuits. Nature Rev. Genet. 10, 859–871 (2009)

    CAS  Article  Google Scholar 

  10. 10

    Grilly, C., Stricker, J., Pang, W., Bennett, M. & Hasty, J. A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae . Mol. Syst. Biol. 3, 127 (2007)

    Article  Google Scholar 

  11. 11

    Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli . Nature 403, 339–342 (2000)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Lu, T. & Collins, J. Dispersing biofilms with engineered enzymatic bacteriophage. Proc. Natl Acad. Sci. USA 104, 11197–11202 (2007)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Friedland, A. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Danino, T., Mondragon-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Tamsir, A., Tabor, J. & Voigt, C. Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’. Nature 469, 212–215 (2011)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Tabor, J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009)

    Article  Google Scholar 

  18. 18

    Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Mondragon-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Entrainment of a population of synthetic genetic oscillators. Science 333, 1315–1319 (2011)

    ADS  MathSciNet  CAS  Article  Google Scholar 

  20. 20

    Tigges, M., Marquez-Lago, T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Westinghouse, G. System of electrical distribution. US patent 373. 035 (1887)

  22. 22

    Lewandowski, W., Azoubib, J. & Klepczynski, W. GPS: primary tool for time transfer. Proc. IEEE 87, 163–172 (1999)

    ADS  Article  Google Scholar 

  23. 23

    Vladimirov, A., Kozyreff, G. & Mandel, P. Synchronization of weakly stable oscillators and semiconductor laser arrays. Europhys. Lett. 61, 613 (2003)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Gast, T. Sensors with oscillating elements. J. Phys. E: Sci. Instrum. 18, 783 (1985)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Ozbudak, E. M., Thattai, M., Kurtser, I., Grossman, A. D. & van Oudenaarden, A. Regulation of noise in the expression of a single gene. Nature Genet. 31, 69–73 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Golding, I., Paulsson, J., Zawilski, S. & Cox, E. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Blake, W. et al. Phenotypic consequences of promoter-mediated transcriptional noise. Mol. Cell 24, 853–865 (2006)

    CAS  Article  Google Scholar 

  29. 29

    Austin, D. et al. Gene network shaping of inherent noise spectra. Nature 439, 608–611 (2006)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Waters, C. & Bassler, B. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005)

    CAS  Article  Google Scholar 

  31. 31

    Ferry, M., Razinkov, I. & Hasty, J. Microfluidics for synthetic biology from design to execution. Methods Enzymol. 497, 295 (2011)

    CAS  Article  Google Scholar 

  32. 32

    Messner, K. & Imlay, J. The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli . J. Biol. Chem. 274, 10119–10128 (1999)

    CAS  Article  Google Scholar 

  33. 33

    Bose, J. L. et al. Bioluminescence in Vibrio fischeri is controlled by the redox-responsive regulator arca. Mol. Microbiol. 65, 538–553 (2007)

    CAS  Article  Google Scholar 

  34. 34

    Georgellis, D., Kwon, O. & Lin, E. Quinones as the redox signal for the arc two-component system of bacteria. Science 292, 2314–2316 (2001)

    CAS  Article  Google Scholar 

  35. 35

    Seaver, L. & Imlay, J. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli . J. Bacteriol. 183, 7182–7189 (2001)

    CAS  Article  Google Scholar 

  36. 36

    Fridovich, I. The biology of oxygen radicals. Science 201, 875–880 (1978)

    ADS  CAS  Article  Google Scholar 

  37. 37

    McCord, J. & Fridovich, I. Superoxide dismutase. J. Biol. Chem. 244, 6049–6055 (1969)

    CAS  PubMed  Google Scholar 

  38. 38

    Berg, J., Tymoczko, J. L. & Stryer, L. Biochemistry (W.H. Freeman, 2006)

    Google Scholar 

  39. 39

    Remington, S. Fluorescent proteins: maturation, photochemistry and photophysics. Curr. Opin. Struct. Biol. 16, 714–721 (2006)

    CAS  Article  Google Scholar 

  40. 40

    Kelner, M., Bagnell, R. & Welch, K. Thioureas react with superoxide radicals to yield a sulfhydryl compound. explanation for protective effect against paraquat. J. Biol. Chem. 265, 1306–1311 (1990)

    CAS  PubMed  Google Scholar 

  41. 41

    Touati, D., Jacques, M., Tardat, B., Bouchard, L. & Despied, S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase. J. Bacteriol. 177, 2305–2314 (1995)

    CAS  Article  Google Scholar 

  42. 42

    Kohanski, M. A., DePristo, M. A. & Collins, J. J. Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol. Cell 37, 311–320 (2010)

    CAS  Article  Google Scholar 

  43. 43

    Nordstrom, D. Worldwide occurrences of arsenic in ground water. Science 296, 2143 (2002)

    CAS  Article  Google Scholar 

  44. 44

    van der Meer, J. & Belkin, S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Rev. Microbiol. 8, 511–522 (2010)

    CAS  Article  Google Scholar 

  45. 45

    Daunert, S. et al. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem. Rev. 100, 2705–2738 (2000)

    CAS  Article  Google Scholar 

  46. 46

    Leveau, J. & Lindow, S. Bioreporters in microbial ecology. Curr. Opin. Microbiol. 5, 259–265 (2002)

    Article  Google Scholar 

  47. 47

    Mather, W., Bennett, M., Hasty, J. & Tsimring, L. Delay-induced degrade-and-fire oscillations in small genetic circuits. Phys. Rev. Lett. 102, 068105 (2009)

    ADS  Article  Google Scholar 

  48. 48

    Quan, J. & Tian, J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE 4, e6441 (2009)

    ADS  Article  Google Scholar 

  49. 49

    Stocker, J. et al. Development of a set of simple bacterial biosensors for quantitative and rapid measurements of arsenite and arsenate in potable water. Environ. Sci. Technol. 37, 4743–4750 (2003)

    ADS  CAS  Article  Google Scholar 

  50. 50

    Keiler, K., Waller, P. & Sauer, R. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993 (1996)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health and General Medicine (R01GM69811), the San Diego Center for Systems Biology (P50GM085764), the DoD NDSEG (A.P.) and NSF Graduate Research (P.S.) Fellowship Programs. L.T. was supported, in part, by the Office of Naval Research (MURI N00014-07-0741). We would like to thank J. Imlay, S. Ali and J. Collins for helpful discussions and J. Hickman, B. Taylor and K. Lomax for their help with illustrations.

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Authors

Contributions

All authors contributed extensively to the work presented in this paper. A.P. and P.S. are equally contributing first authors.

Corresponding author

Correspondence to Jeff Hasty.

Supplementary information

Supplementary Information

The file contains Supplementary Text and Data, Supplementary Figures 1-12 with legends and additional references. (PDF 1384 kb)

Supplementary Movie 1

The movie shows timelapse fluorescence microscopy of a 200 trap sensor array displaying NDH-2 engineered synchronization. An EMCCD camera was used to keep exposure times extremely low (4X magnification, 20ms, 95% attenuation) to ensure no fluorescence interaction, hence the appearance of lower signal. (MOV 11321 kb)

Supplementary Movie 2

The movie shows timelapse fluorescence microscopy of the 500 trap biosensor array showing the onset of synchronization from disparate initial conditions using period modulator circuit. Flashes indicate changes in arsenite concentration which result in changes in the oscillatory period. (MOV 9624 kb)

Supplementary Movie 3

The movie shows timelapse fluorescence microscopy of a sensor array containing thresholding circuit. Red color indicates addition of 0.25 mM arsenite that initiates oscillations in blue. (MOV 1671 kb)

Supplementary Movie 4

The movie shows timelapse fluorescence microscopy of a modified 500 trap sensor array in which traps are farther apart. This increased separation results in anti phase oscillations, where a biopixel and its nearest neighbors alternate bursts. (MOV 2678 kb)

Supplementary Movie 5

The movie shows timelapse fluorescence microscopy of the 12,000 trap scaled up array showing oscillation and synchronization maintained over a maximum distance of 27 mm. (MOV 7711 kb)

Supplementary Movie 6

The movie shows Real time microscopy depicting the loading of our microfluidic device. Cells flow in from the cell port and fill the trapping regions. (MOV 5442 kb)

Supplementary Movie 7

The movie shows Timelapse fluorescence microscopy of a modified 500 trap sensor array in which traps of 2 sizes are present. This results in 2:1 resonant oscillations where larger traps oscillate at twice the frequency of smaller traps. (MOV 2456 kb)

Supplementary Movie 8

The movie shows timelapse fluorescence microscopy of a 500 trap sensor array showing unsynchronized oscillations when NDH-2 is not present and high-intensity fluorescence bursts are not used. (MOV 10904 kb)

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Prindle, A., Samayoa, P., Razinkov, I. et al. A sensing array of radically coupled genetic ‘biopixels’. Nature 481, 39–44 (2012). https://doi.org/10.1038/nature10722

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