Anne Meyer1
University of Rochester1
3D printing of engineered bacteria is an evolving field that employs the tools of synthetic biology to express desired proteins and peptides while maintaining control over the shape and spatial organization of the resultant biomaterials. Previous bacterial 3D printers have been limited to producing low aspect ratio prints that become unstable and collapse under their own weight at greater heights. By leveraging open-source hardware and software, we have designed and modified a low-cost, commercial 3D printer into a bacterial bioprinter that can print complex, free-standing structures with high aspect ratios. To print complex structures with sharply defined edges, a novel syringe mount was designed with retraction functionality that pauses bio-ink extrusion during non-printing periods. Our new printer deposits bio-ink within gel slurries with viscoplastic properties, allowing delivery of bio-ink gellification agents immediately upon printing. This technique allows us to print stable, high aspect ratio (15:1), free-standing structures with sharper resolutions and internal cavities. A challenge to bioprinting is providing a supply of fresh nutrients and exchanging waste byproducts to support the health of the embedded microbes without damaging the printed structures. To address this, we developed a method to incubate the bioprints within a stable agarose-based gel slurry that permits diffusion of nutrients. Bioprints of engineered <i>Escherichia coli</i> strains incubated within a nutrient-infused slurry for seven days demonstrated 100-times higher colony-forming units compared to bioprints incubated without added nutrients, suggesting that nutrients diffuse through the slurry to increase viability of bacteria within bioprints. Printing complex structures by combining a low-cost 3D printer with a nutrient delivery method will enable the printing of complex biomaterials with extended functional lifespans and novel applications, including enzymatic degradation, multifunctional sensors, biofilm applications, and more.