Macroscopic De-Novo Engineered Living Materials

Engineered living materials (ELM) are a new class of materials that contain living cells and thus have some of the unique capabilities of living systems, e.g. autonomous growth, self-repairing, or the ability to sense and respond to the environment. Most existing examples of macroscopic ELMs have introduced novel properties onto naturally-occurring biological materials. However, programming macroscopic material formation de novo from a model organism would offer much greater, user-defined control over material structure and function. We created a bottom-up designed ELM by engineering Caulobacter crescentus to display weakly interacting peptides at high density on the cell surface. This enabled the self-assembly of a novel, macroscopic ELM. We included the SpyTag peptide into the displayed protein, allowing for user-defined functionalization via SpyTag-SpyCatcher interactions. As C. crescentus expressing the synthetic construct grows, the cells assemble into a pellicle at the air-water interface. This layer expands in thickness and ultimately sinks into the media to form free-floating, macroscopic materials shaped as cocoons. As expected, the cocoons are predominantly composed of cells bound to each other through the synthetic protein. Surprisingly, we also observed the synthetic proteins dissociate from the cell surface to form long ~2 micron-thick filaments, which bundle into ~100-micron-thick fibers running throughout the cocoon. The morphology and size of these cocoons can be tuned by changing physical parameters, such as shaking speed and media volume. Within a cocoon, C. crescentus cells remain viable and can re-seed growth of new cocoons even after desiccation. Cocoons can also be processed into different 3D shapes and can be mixed with inorganic materials to form inorganic-organic composites. Taken together, this work provides design rules and a technological platform for programming bottom-up assembly of cells into hierarchically-structured, multi-functional living materials.