Resilient living materials built by printing bacterial spores

Abstract

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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Fig. 1: 3D printer design and development of the bioink components.
Fig. 2: 3D printing of living materials.
Fig. 3: Cutting the object induces germination of spores.
Fig. 4: Induction of genome-encoded sensors in the printed material.
Fig. 5: Resilience of living materials printed with B. subtilis spores.
Fig. 6: Printing a wound-shaped hydrogel containing bacteria that respond to S. aureus.

Data availability

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.

Code availability

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.

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Acknowledgements

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.

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L.M.G designed and built the printer and performed the materials and genetic engineering experiments. N.M. built the AIP and lysostaphin strains and aided in the construction and analysis of those materials. C.A.V. and L.M.G. conceived of the project, analyzed the data, and wrote the manuscript.

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Correspondence to Christopher A. Voigt.

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Supplementary Tables 1–8, Supplementary Figs. 1–45, Supplementary Notes 1–2.

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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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