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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.

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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.

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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.

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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

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.

Correspondence to Chao Zhong.

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Supplementary Information

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

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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.