Caroline Ajo-Franklin1 Marimikel Charrier1 Dong Li1 Sneha Jani1 Victor Mann1 Behzad Rad1 Bruce Cohen1 Kathleen Ryan2 Paul Ashby1

1, Lawrence Berkeley National Laboratory, Berkeley, California, United States
2, Dept of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States

Structural materials synthesized by organisms, such as bones, shells, and wood, exhibit remarkable mechanical properties due to their hierarchical assembly of hard and soft components across the nanometer to the micron scales. While engineering analogs to these materials would open new frontiers, there is currently no route to mimic the 2D hierarchical ordering of natural composites. Here we lay the foundations for bottom-up assembly of engineered living-material composites along the cell body using a synthetic biology approach and demonstrate the hierarchical assembly of these composites confers switchable mechanical properties. We engineer the surface-layer (S-layer) of Caulobacter crescentus to display peptides that permit covalent attachment of proteins, nanocrystals, and protein-based polymers to the extracellular surface without additional engineering. This cell surface binding is uniform, specific, and covalent, and its density can be controlled based on the location of the insertion within the S-layer. Using this platform, we construct composite materials composed of living C. crescentus cells crosslinked by nanocrystals. These ‘hybrid bacterial spheroids’ are 30 times more stiff than cell-nanocrystal composites lacking these crosslinks. Additionally, the stiffness of these composites can be changed dynamically by breaking the cell-nanocrystal crosslinking. Taken together, this work provides a platform for creating hierarchically-assembled living materials with switchable mechanical properties.