Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Programmable and printable Bacillus subtilis biofilms as engineered living materials

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 options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Design for a programmable and printable B. subtilis biofilm production platform.
Fig. 2: Extracellular secretion and assembly of TasA-R amyloid proteins into fibrous networks associated with the surfaces of engineered B. subtilis cells.
Fig. 3: Functional characterization of engineered B. subtilis biofilms.
Fig. 4: Morphology, rheological properties, and 3D printing of engineered B. subtilis biofilms.
Fig. 5: Self-regeneration, biofabrication, long-term viability, and functional performance of hydrogel-trapped B. subtilis biofilms.

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.

References

  1. 1.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  3. 3.

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

    Article  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

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

    CAS  Article  PubMed  Google Scholar 

  13. 13.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  15. 15.

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

    Article  CAS  Google Scholar 

  16. 16.

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

    Article  CAS  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

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

    CAS  Article  PubMed  Google Scholar 

  30. 30.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  36. 36.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  38. 38.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Chao Zhong.

Ethics declarations

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.

Additional information

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

Supplementary information

Supplementary Information

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

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, J., Liu, S., Zhang, C. et al. Programmable and printable Bacillus subtilis biofilms as engineered living materials. Nat Chem Biol 15, 34–41 (2019). https://doi.org/10.1038/s41589-018-0169-2

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing