Assessing Fabrication Strategies for Robust Bacterial Confinement and Protein Release in Alginate-Based Engineered Living Materials

High Resolution Image Download MS PowerPoint Slide In engineered living materials (ELMs) made of genetically engineered bacteria embedded in polymeric matrices, it is desirable for the bacteria to remain contained for biosafety and reliable performance. Such ELMs are being explored for a variety of applications, including smart drug delivery, in which the material also needs to allow protein release. Achieving this balance requires materials that simultaneously support cell viability, prevent bacterial escape, and permit controlled molecular diffusion. Here, we systematically dissect how alginate cross-linking chemistry, buffer composition, and secondary network reinforcement shape the performance of core–shell beads encapsulating Escherichia coli Nissle 1917 engineered to secrete a model protein. We identify a 4 h Ca 2+ cross-linking window as the upper limit that maintains robust core viability and week-long confinement. For improved confinement, additional cross-linking strategies, including glutaraldehyde, polyethylenimine, and tannic acid, were tested and found to enhance the shell’s barrier properties to prevent leakage for up to 15 days but also negatively impacted protein release. Finally, replacing Ca 2+ with Ba 2+ as the cross-linking divalent cation yields the most effective solution, producing beads with exceptional long-term bacterial confinement capability (>20 days) while allowing for comparable protein secretion levels to Ca 2+ controls. Together, these results establish a a materials-centric design framework for fabricating alginate-based ELMs that achieve durable confinement without compromising functional protein output and highlight barium-alginate shells as a promising platform for fabricating ELMs toward therapeutic applications.