Materials can be made multifunctional by embedding them with living cells that perform sensing, synthesis, energy production, and physical movement. A challenge is that the conditions needed for living cells are not conducive to materials processing and require continuous water and nutrients. Here, we present a three dimensional (3D) printer that can mix material and cell streams to build 3D objects. Bacillus subtilis spores were printed within the material and germinated on its exterior surface, including spontaneously in new cracks. The material was resilient to extreme stresses, including desiccation, solvents, osmolarity, pH, ultraviolet light, and γ-radiation. Genetic engineering enabled the bacteria to respond to stimuli or produce chemicals on demand. As a demonstration, we printed custom-shaped hydrogels containing bacteria that can sense or kill Staphylococcus aureus, a causative agent of infections. This work demonstrates materials endued with living functions that can be used in applications that require storage or exposure to environmental stresses.
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The data that supports the findings of this study are available from the corresponding author on request. Plasmids and Bacilli strains are available from Addgene and BGSC, respectively.
All of the Arduino scripts are available privately from GitHub after requesting permission from the authors using the following link: https://github.com/linagonzalez87/3D-printer-Cell-Line.git.
Nguyen, P. Q., Courchesne, N.-M. 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, 1704847 (2018).
Chen, A. Y., Zhong, C. & Lu, T. K. Engineering living functional materials. ACS Synth. Biol. 4, 8–11 (2015).
Balasubramanian, S., Aubin-Tam, M.-E. & Meyer, A. S. 3D printing for the fabrication of biofilm-based functional living materials. ACS Synth. Biol. 8, 1564–1567 (2019).
Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A. & Lewis, J. A. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl Acad. Sci. USA 113, 3179 (2016).
Mao, A. S. & Mooney, D. J. Regenerative medicine: current therapies and future directions. Proc. Natl Acad. Sci. USA 112, 14452–14459 (2015).
Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J. & Voigt, C. A. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat. Chem. Biol. 15, 196–204 (2019).
Gerber, L. C., Koehler, F. M., Grass, R. N. & Stark, W. J. Incorporating microorganisms into polymer layers provides bioinspired functional living materials. Proc. Natl Acad. Sci. USA 109, 90–94 (2012).
Ball, P. Synthetic biology—engineering nature to make materials. MRS Bull. 43, 477–484 (2018).
Jonkers, H. M., Thijssen, A., Muyzer, G., Copuroglu, O. & Schlangen, E. Application of bacteria as self-healing agent for the development of sustainable concrete. Ecol. Eng. 36, 230–235 (2010).
Chen, X., Mahadevan, L., Driks, A. & Sahin, O. Bacillus spores as building blocks for stimuli-responsive materials and nanogenerators. Nat. Nanotechnol. 9, 137–141 (2014).
Zhang, C. et al. Engineered Bacillus subtilis biofilms as living glues. Mater. Today 28, 40–48 (2019).
Haneef, M. et al. Advanced materials from fungal mycelium: fabrication and tuning of physical properties. Sci. Rep. 7, 41292 (2017).
Wang, W. et al. Harnessing the hygroscopic and biofluorescent behaviors of genetically tractable microbial cells to design biohybrid wearables. Sci. Adv. 3, e1601984 (2017).
Chen, A. Y. et al. Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515–523 (2014).
Cao, Y. et al. Programmable assembly of pressure sensors using pattern-forming bacteria. Nat. Biotechnol. 35, 1087 (2017).
Charrier, M. et al. Engineering the S-Layer of Caulobacter crescentus as a foundation for stable, high-density, 2D living materials. ACS Synth. Biol. 8, 181–190 (2019).
Ngo, T. D., Kashani, A., Imbalzano, G., Nguyen, K. T. Q. & Hui, D. Additive manufacturing (3D printing): a review of materials, methods, applications and challenges. Compos. Part B-Eng. 143, 172–196 (2018).
Keating, S. J. et al. 3D printed multimaterial microfluidic valve. PLoS One 11, e0160624 (2016).
Schmieden, D. T. et al. Printing of patterned, engineered E. coli biofilms with a low-cost 3D printer. ACS Synth. Biol. 7, 1328–1337 (2018).
Connell, J. L., Ritschdorff, E. T., Whiteley, M. & Shear, J. B. 3D printing of microscopic bacterial communities. Proc. Natl Acad. Sci. USA 110, 18380 (2013).
Liu, X. et al. 3D Printing of living responsive materials and devices. Adv. Mater. 30, 1704821 (2017).
Lehner, B. A. E., Schmieden, D. T. & Meyer, A. S. A straightforward approach for 3D bacterial printing. ACS Synth. Biol. 6, 1124–1130 (2017).
Schaffner, M., Rühs, P. A., Coulter, F., Kilcher, S. & Studart, A. R. 3D printing of bacteria into functional complex materials. Sci. Adv. 3, eaao6804 (2017).
Huang, J. et al. Programmable and printable Bacillus subtilis biofilms as engineered living materials. Nat. Chem. Biol. 15, 34–41 (2019).
McKenney, P. T., Driks, A. & Eichenberger, P. The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat. Rev. Microbiol. 11, 33 (2012).
Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 101, 514–525 (2006).
Moeller, R. et al. Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment. J. Bacteriol. 190, 1134 (2008).
Setlow, B., Atluri, S., Kitchel, R., Koziol-Dube, K. & Setlow, P. Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective α/β-type small acid-soluble proteins. J. Bacteriol. 188, 3740–3747 (2006).
Sahin, O., Yong, E. H., Driks, A. & Mahadevan, L. Physical basis for the adaptive flexibility of Bacillus spore coats. J. R. Soc. Interface 9, 3156–3160 (2012).
Cano, R. J. & Borucki, M. K. Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. Science 268, 1060 (1995).
Ulrich, N. et al. Experimental studies addressing the longevity of Bacillus subtilis spores–the first data from a 500-year experiment. PLoS One 13, e0208425 (2018).
Cutting, S. M., Hong, H. A., Baccigalupi, L. & Ricca, E. Oral vaccine delivery by recombinant spore probiotics. Int. Rev. Immunol. 28, 487–505 (2009).
Yung, P. T. & Ponce, A. Fast sterility assessment by germinable-endospore biodosimetry. Appl. Environ. Microbiol. 74, 7669–7674 (2008).
N. Turner, B., Strong, R. & A. Gold, S. A review of melt extrusion additive manufacturing processes: I. Process design and modeling. Rapid Prototyp. J. 20, 192–204 (2014).
Bose, B. & Grossman, A. D. Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease. J. Bacteriol. 193, 22 (2011).
Miwa, Y., Nakata, A., Ogiwara, A., Yamamoto, M. & Fujita, Y. Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis. Nucleic Acids Res. 28, 1206–1210 (2000).
Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J. & Setlow, P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548 (2000).
Harrell, C. R., Djonov, V., Fellabaum, C. & Volarevic, V. Risks of using sterilization by gamma radiation: the other side of the coin. Int. J. Med. Sci. 15, 274–279 (2018).
Lee, A. S. et al. Methicillin-resistant Staphylococcus aureus. Nat. Rev. Dis. Prim. 4, 18033 (2018).
Awadhiya, A., Tyeb, S., Rathore, K. & Verma, V. Agarose bioplastic-based drug delivery system for surgical and wound dressings. Eng. Life Sci. 17, 204–214 (2017).
Jensen, R. O., Winzer, K., Clarke, S. R., Chan, W. C. & Williams, P. Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC. J. Mol. Biol. 381, 300–309 (2008).
Johnson, C. T. et al. Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.Proc. Natl Acad. Sci. USA 115, E4960–E4969 (2018).
Wieland Brown, L. C., Acker, M. G., Clardy, J., Walsh, C. T. & Fischbach, M. A. Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin. Proc. Natl Acad. Sci. USA 106, 2549 (2009).
Acker, M. G., Bowers, A. A. & Walsh, C. T. Generation of thiocillin variants by prepeptide gene replacement and in vivo processing by Bacillus cereus. J. Am. Chem. Soc. 131, 17563–17565 (2009).
Cámara, M., Hardman, A., Williams, P. & Milton, D. Quorum sensing in Vibrio cholerae. Nat. Genet. 32, 217–218 (2002).
Rutherford, S. T. & Bassler, B. L. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb. Perspect. Med. 2, a012427 (2012).
Hwang, I. Y. et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8, 15028 (2017).
Thoendel, M., Kavanaugh, J. S., Flack, C. E. & Horswill, A. R. Peptide signaling in the Staphylococci. Chem. Rev. 111, 117–151 (2011).
Koenig, R. L., Ray, J. L., Maleki, S. J., Smeltzer, M. S. & Hurlburt, B. K. Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region. J. Bacteriol. 186, 7549 (2004).
Kokai-Kun, J. F., Walsh, S. M., Chanturiya, T. & Mond, J. J. Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrob. Agents Chemother. 47, 1589–1597 (2003).
Bayer, T. S. & Samson, J. A. Bacterial methods. US patent 9023635B2 (2015).
Hwang, I. Y. et al. Engineering microbes for targeted strikes against human pathogens. Cell. Mol. Life Sci. 75, 2719–2733 (2018).
L.M.G. and C.A.V. were supported by the Vannevar Bush Faculty Fellowship program sponsored by the Basic Research Office of the Assistant Secretary of Defense for Research and Engineering and funded by the Office of Naval Research through grant number N00014-16-1-2509. This research was also funded by the Institute for Collaborative Biotechnologies through contract number W911NF-09-0001 with the U.S. Army Research Office. We also thank the Koch Institute Swanson Biotechnology Center for technical support, notably the Nanotechnology Materials Core Center. We thank Christopher Walsh at Harvard Medical School for providing the thiocillin-producing cells.
The authors declare no competing interests.
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González, L.M., Mukhitov, N. & Voigt, C.A. Resilient living materials built by printing bacterial spores. Nat Chem Biol 16, 126–133 (2020). https://doi.org/10.1038/s41589-019-0412-5
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