Cohesive Living Bacterial Films with Tunable Mechanical Properties from Cell Surface Protein Display
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1.
California Institute of Technology
Abstract
Engineered living materials (ELMs) constitute a novel class of functional materials that contain living organisms. The mechanical properties of many such systems are dominated by the polymeric matrices used to encapsulate the cellular components of the material, making it hard to tune the mechanical behavior through genetic manipulation. To address this issue, we have developed living materials in which mechanical properties are controlled by the cell-surface display of engineered proteins. Here, we show that engineered Esherichia coli cells outfitted with surface-displayed elastin-like proteins (ELPs, designated E6) grow into soft, cohesive bacterial films with biaxial moduli around 14 kPa. When subjected to bulge-testing, such films yielded at strains of approximately 10%. Introduction of a single cysteine residue near the exposed N-terminus of the ELP (to afford a protein designated CE6) increases the film modulus 3-fold to 44 kPa and eliminates the yielding behavior. When subjected to oscillatory stress, films prepared from E. coli strains bearing CE6 exhibit modest hysteresis and full strain recovery; in E6 films much more significant hysteresis and substantial plastic deformation are observed. CE6 films heal autonomously after damage, with the biaxial modulus fully restored after a few hours. This work establishes an approach to living materials with genetically programmable mechanical properties and a capacity for self-healing. Such materials may find application in biomanufacturing, biosensing, and bioremediation.
Copyright and License
Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0 .
Acknowledgement
This work was supported by the DARPA Engineered Living Materials program (award # HR0011-17-2-0037) and by the Army Research Office through the Institute for Collaborative Biotechnologies (award # W911NF-19-2-0026). The results reported herein are described in the doctoral theses of Hanwei Liu (“Engineered Living Material Based on Protein-Mediated Bacterial Assembly,” California Institute of Technology, 2024) and Priya Chittur (“A Millifluidic Bulge Test for Multiscale Properties of Engineered Biofilms,” California Institute of Technology, 2023). We thank Dr Andres Collazo for assistance with some of the preliminary data obtained by confocal microscopy in the Biological Imaging Facility of the Caltech Beckman Institute, which is supported by the Arnold and Mabel Beckman Foundation. We thank Dr Dennis Ko and Rohit Srikanth for performing cycles of infusion and withdrawal pump experiments.
Contributions
H.L. and P.K.C. contributed equally to this work. H.L. conceived the methods used for the preparation of bacterial films under the supervision of D.A.T. H.L. was responsible for all genetic engineering experiments, bacterial film growth, sample preparation, and fluorescence imaging. P.K.C. performed all experiments related to the biofilm mechanical property analysis using the bulge test device under the supervision of J.A.K. This paper was written by H.L.; P.K.C. provided written material characterization methods and values of material properties. All authors contributed to the editing of the final manuscript.
Supplemental Material
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00528.
- Detailed description of Videos S1–S5 (PDF)
- Additional experimental information including tables of plasmid constructs and protein sequences, additional microscopy images, Western blotting and mass spectrometry data, and bulge test device setup and image analysis details (PDF)
- Videos of OCT-scanned “Ramp” bulge tests of E6-AT and CE6-AT films, peeling experiments, and the only observed failure event of a CE6-AT film (ZIP)
Additional Information
Published as part of ACS Synthetic Biology special issue “Materials Design by Synthetic Biology”.
Files
Additional details
- Defense Advanced Research Projects Agency
- HR0011-17-2-0037
- Institute for Collaborative Biotechnologies
- W911NF-19-2-0026
- Available
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2024-11-01Published online
- Publication Status
- Published