Abstract

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The main data supporting the findings of this study are available within the article and its Supplementary Information files. Extra data are available from the corresponding author upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Chen, A. Y. et al. Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515–523 (2014).

  2. 2.

    Nguyen, P. Q., Courchesne, N. D., Duraj-Thatte, A., Praveschotinunt, P. & Joshi, N. S. Engineered living materials: prospects and challenges for using biological systems to direct the assembly of smart materials. Adv. Mater. 30, e1704847 (2018).

  3. 3.

    Ball, P. Synthetic biology—engineering nature to make materials. MRS Bull. 43, 477–484 (2018).

  4. 4.

    Wang, Y., Pu, J., An, B., Lu, T. K. & Zhong, C. Emerging paradigms for synthetic design of functional amyloids. J. Mol. Biol. 430, 3720–3734 (2018).

  5. 5.

    Tallawi, M., Opitz, M. & Lieleg, O. Modulation of the mechanical properties of bacterial biofilms in response to environmental challenges. Biomater. Sci. 5, 887–900 (2017).

  6. 6.

    Vlamakis, H., Chai, Y., Beauregard, P., Losick, R. & Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nat. Rev. Microbiol. 11, 157–168 (2013).

  7. 7.

    Barnhart, M. M. & Chapman, M. R. Curli biogenesis and function. Annu. Rev. Microbiol. 60, 131–147 (2006).

  8. 8.

    DeBenedictis, E. P., Liu, J. & Keten, S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Sci. Adv. 2, e1600998 (2016).

  9. 9.

    Edwards, S. J. & Kjellerup, B. V. Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals. Appl. Microbiol. Biotechnol. 97, 9909–9921 (2013).

  10. 10.

    Donato, V. et al. Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway. Nat. Commun. 8, 14332 (2017).

  11. 11.

    Hobley, L., Harkins, C., MacPhee, C. E. & Stanley-Wall, N. R. Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes. FEMS Microbiol. Rev. 39, 649–669 (2015).

  12. 12.

    Yates, M. D. et al. Measuring conductivity of living Geobacter sulfurreducens biofilms. Nat. Nanotechnol. 11, 910–913 (2016).

  13. 13.

    Cao, Y. et al. Programmable assembly of pressure sensors using pattern-forming bacteria. Nat. Biotechnol. 35, 1087–1093 (2017).

  14. 14.

    Nguyen, P. Q., Botyanszki, Z., Tay, P. K. & Joshi, N. S. Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945 (2014).

  15. 15.

    Wang, X. et al. Programming cells for dynamic assembly of inorganic nano-objects with spatiotemporal control. Adv. Mater. 30, 1705968 (2018).

  16. 16.

    Liu, X. et al. 3D printing of living responsive materials and devices. Adv. Mater. 30, 1704821 (2018).

  17. 17.

    Liu, X. et al. Stretchable living materials and devices with hydrogel-elastomer hybrids hosting programmed cells. Proc. Natl Acad. Sci. USA 114, 2200–2205 (2017).

  18. 18.

    Liu, L. et al. Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology. Appl. Microbiol. Biotechnol. 97, 6113–6127 (2013).

  19. 19.

    Cairns, L. S., Hobley, L. & Stanley-Wall, N. R. Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms. Mol. Microbiol. 93, 587–598 (2014).

  20. 20.

    Kesel, S. et al. Direct comparison of physical properties of Bacillus subtilis NCIB 3610 and B-1 biofilms. Appl. Environ. Microbiol. 82, 2424–2432 (2016).

  21. 21.

    Driks, A. Tapping into the biofilm: insights into assembly and disassembly of a novel amyloid fibre in Bacillus subtilis. Mol. Microbiol. 80, 1133–1136 (2011).

  22. 22.

    Diehl, A. et al. Structural changes of TasA in biofilm formation of Bacillus subtilis. Proc. Natl Acad. Sci. USA 115, 3237–3242 (2018).

  23. 23.

    Chai, L. et al. Isolation, characterization, and aggregation of a structured bacterial matrix precursor. J. Biol. Chem. 288, 17559–17568 (2013).

  24. 24.

    Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).

  25. 25.

    Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).

  26. 26.

    Botyanszki, Z., Tay, P. K., Nguyen, P. Q., Nussbaumer, M. G. & Joshi, N. S. Engineered catalytic biofilms: site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnol. Bioeng. 112, 2016–2024 (2015).

  27. 27.

    Slavin, Y. N., Asnis, J., Häfeli, U. O. & Bach, H. Metal nanoparticles: understanding the mechanisms behind antibacterial activity. J. Nanobiotechnology 15, 65 (2017).

  28. 28.

    Schaffner, M., Ruhs, P. A., Coulter, F., Kilcher, S. & Studart, A. R. 3D printing of bacteria into functional complex materials. Sci. Adv. 3, eaao6804 (2017).

  29. 29.

    Grumbein, S. et al. Hydrophobic properties of biofilm-enriched hybrid mortar. Adv. Mater. 28, 8138–8143 (2016).

  30. 30.

    Lieleg, O., Caldara, M., Baumgärtel, R. & Ribbeck, K. Mechanical robustness of Pseudomonas aeruginosa biofilms. Soft Matter 7, 3307–3314 (2011).

  31. 31.

    Luo, Y., Lode, A. & Gelinsky, M. Direct plotting of three-dimensional hollow fiber scaffolds based on concentrated alginate pastes for tissue engineering. Adv. Healthc. Mater. 2, 777–783 (2013).

  32. 32.

    Connell, J. L., Ritschdorff, E. T., Whiteley, M. & Shear, J. B. 3D printing of microscopic bacterial communities. Proc. Natl Acad. Sci. USA 110, 18380–18385 (2013).

  33. 33.

    Orive, G., Tam, S. K., Pedraz, J. L. & Hallé, J. P. Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy. Biomaterials. 27, 3691–3700 (2006).

  34. 34.

    Xue, S. et al. A synthetic-biology-inspired therapeutic strategy for targeting and treating hepatogenous diabetes. Mol. Ther. 25, 443–455 (2017).

  35. 35.

    Wilking, J. N., Angelini, T. E., Seminara, A., Brenner, M. P. & Weitz, D. A. Biofilms as complex fluids. MRS Bull. 36, 385–391 (2011).

  36. 36.

    Flemming, H. C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).

  37. 37.

    Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. & Stuber, D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat. Biotechnol. 6, 1321–1325 (1988).

  38. 38.

    Zhong, C. et al. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat. Nanotechnol. 9, 858–866 (2014).

  39. 39.

    Arakaki, A., Webb, J. & Matsunaga, T. A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1. J. Biol. Chem. 278, 8745–8750 (2003).

  40. 40.

    Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

  41. 41.

    McEvoy, A. L. et al. mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities. PLoS ONE. 7, e51314 (2012).

  42. 42.

    Lu, H. D., Wheeldon, I. R. & Banta, S. Catalytic biomaterials: engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels. Protein Eng. Des. Sel. 23, 559–566 (2010).

  43. 43.

    Konkol, M. A., Blair, K. M. & Kearns, D. B. Plasmid-encoded ComI inhibits competence in the ancestral 3610 strain of Bacillus subtilis. J. Bacteriol. 195, 4085–4093 (2013).

  44. 44.

    Arnaud, M., Chastanet, A. & Débarbouillé, M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl. Environ. Microbiol. 70, 6887–6891 (2004).

  45. 45.

    Romero, D., Aguilar, C., Losick, R. & Kolter, R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc. Natl Acad. Sci. USA 107, 2230–2234 (2010).

  46. 46.

    Branda, S. S., González-Pastor, J. E., Ben-Yehuda, S., Losick, R. & Kolter, R. Fruiting body formation by Bacillus subtilis. Proc. Natl Acad. Sci. USA 98, 11621–11626 (2001).

  47. 47.

    Branda, S. S., Chu, F., Kearns, D. B., Losick, R. & Kolter, R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59, 1229–1238 (2006).

  48. 48.

    Arnaouteli, S. et al. Bifunctionality of a biofilm matrix protein controlled by redox state. Proc. Natl Acad. Sci. USA 114, E6184–E6191 (2017).

Download references

Acknowledgements

The authors thank D. Kearns (Indiana University) for the kind gift of the B. subtilis 2569 strain, and Y. Liu for the software instruction. Regular TEM characterization was performed at the National Center for Protein Science, Shanghai. Fluorescence microscopy was performed at the Molecular Imaging Core Facility of SLST, Shanghai Tech University. This work was funded by the Science and Technology Commission of Shanghai Municipality (17JC1403900), National Natural Science Foundation of China (No. 31570972), and 2016 Open Financial Fund of Qingdao National Laboratory for Marine Science and Technology (Grant No. QNLM2016ORP0403) for C. Zhong; C.Zhong. also acknowledges start-up funding support from ShanghaiTech University and 1000 Youth Talents Program, granted by the Chinese Central Government. The work was also partially funded by the National Natural Science Foundation of China (No.31872728) for J.H., and the National Natural Science Foundation of China (NSFC: No. 31522017, No. 31470834, No. 31670869) for H.Y.

Author information

Author notes

  1. These authors contributed equally: Jiaofang Huang, Suying Liu, Chen Zhang.

Affiliations

  1. Materials and Physical Biology Division School of Physical Science and Technology, ShanghaiTech University, Shanghai, China

    • Jiaofang Huang
    • , Suying Liu
    • , Chen Zhang
    • , Xinyu Wang
    • , Jiahua Pu
    • , Tianxin Zhao
    • , Ke Li
    • , Yanyi Wang
    • , Jicong Zhang
    •  & Chao Zhong
  2. Division of Physical Biology and Bioimaging Center Shanghai Synchrotron Radiation Facility CAS Key Laboratory of InterfacialPhysics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China

    • Suying Liu
    • , Lihua Wang
    •  & Chunhai Fan
  3. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    • Chen Zhang
  4. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    • Fang Ba
  5. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    • Shuai Xue
    •  & Haifeng Ye
  6. Shanghai Key Laboratory of Green Chemistry and Chemical Processes School of Chemistry and Molecular Engineering, EastChina Normal University, Shanghai, China

    • Lihua Wang
  7. School of Chemistry and Chemical Engineering, and Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    • Chunhai Fan
  8. Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA

    • Timothy K. Lu

Authors

  1. Search for Jiaofang Huang in:

  2. Search for Suying Liu in:

  3. Search for Chen Zhang in:

  4. Search for Xinyu Wang in:

  5. Search for Jiahua Pu in:

  6. Search for Fang Ba in:

  7. Search for Shuai Xue in:

  8. Search for Haifeng Ye in:

  9. Search for Tianxin Zhao in:

  10. Search for Ke Li in:

  11. Search for Yanyi Wang in:

  12. Search for Jicong Zhang in:

  13. Search for Lihua Wang in:

  14. Search for Chunhai Fan in:

  15. Search for Timothy K. Lu in:

  16. Search for Chao Zhong in:

Contributions

C. Zhong directed the research; J.H. and C. Zhong conceived of the idea and designed the research. J.H., S.L. and C. Zhang did the rheology and instron measurements. S.L., J.H., J.P., and X.W. assayed the MO and PNP degradation. S.L., J.H., F.B. and Y.W. performed the paraoxon degradation, fluorescence experiments and 3D printing. J.H., C. Zhang, T.Z., and K.L. carried out the molecular constructions. J.H., S.L., and J.Z. performed the HPLC assay, S.X., H.Y., and J.H. did the microencapsulation. J.H., S.L., C. Zhang, and C. Zhong analyzed the data, discussed results, and wrote the manuscript, with the help from LW, C.F. and T.K.L.

Competing interests

The authors have filed two provisional patents based on this work (CN/201611156490.X and PCT/CN2018/100538). T.K.L. is a co-founder of Senti Biosciences, Synlogic, Engine Biosciences, TangoTherapeutics, Corvium, BiomX, and Eligo Biosciences. T.K.L. also holds financial interests in nest.bio, Ampliphi,IndieBio, and MedicusTek.

Corresponding author

Correspondence to Chao Zhong.

Supplementary information

  1. Supplementary Information

    Supplementary Tables 1–5, Supplementary Figures 1–20, and Supplementary Notes 1–2

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41589-018-0169-2