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The latter approach yielded remarkably high cell densities of 10 9 cells ml −1, but the need for freeze-drying could significantly affect the survival rate of other microbial species as well as their thixotropic behavior. Also, photo-crosslinked pluronic F127 acrylate-based bioinks have been utilized to print living, responsive materials/devices, and catalytically active living materials 4, 6, 13.Īn alternative strategy made use of freeze-dried Saccharomyces cerevisiae as the primary component of a bioink formulation consisting of nanocellulose, polyethylene glycol dimethacrylate, and a photoinitiator 3. In another approach, a multi-material bioink comprised of hyaluronic acid, κ-carrageenan, fumed silica, and a photo-initiator was employed to 3D-print Pseudomonas putida and Acetobacter xylinum. A similar ionic crosslinking strategy was exploited to generate photocurrent with 3D printed cyanobacteria 12. coli was extruded onto a printing surface consisting of calcium chloride, upon which the alginate molecules crosslink to form a solidified gel 7. In an early example of this concept, a mixture of alginate and E. Although inkjet printing, contact printing, screen printing, and lithographic techniques have been explored to print microbes, extrusion-based bioprinting has become one of the most widely used techniques due to its simplicity, compatibility with a variety of bioinks, and cost-effective instrumentation 2, 9, 10, 11. In this work, we present the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures.ģD bioprinting technology, which is relatively well-established for printing mammalian cells in the context of tissue engineering, has more recently been applied to print microbial cells for biotechnological and biomedical applications 1, 2, 3, 4, 5, 6, 7, 8. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics, and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli ( E. Here we set out to develop a bioink, termed as “microbial ink” that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. The emerging field of living materials has leveraged microbial engineering to produce materials for various applications but building 3D structures in arbitrary patterns and shapes has been a major challenge. Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions.