Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing Biological Engineering Biotechnology Bacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial bacteriocins are alternatives to conventional antibiotics and are characterized by unique cell-killing mechanisms, including pore formation on cell membranes, nuclease activity, and cell wall synthesis inhibition. Here, we used cell-free protein synthesis to rapidly evaluate the antibiofilm activities of colicins E1, E2, and E3. We found that E2 (with DNase activity) most effectively killed target biofilm cells (i.e., the K361 strain) while leaving nontargeted biofilms intact. We then engineered probiotic Escherichia coli microorganisms with genetic circuits to controllably synthesize and secrete colicin E2, which successfully inhibited biofilms and killed preformed indicator biofilms. Our findings suggest that colicins rapidly and selectively kill target biofilm cells in multispecies biofilms and demonstrate the potential of using microorganisms engineered to produce antimicrobial colicin proteins as live therapeutic strategies to treat biofilm-associated infections. Sponsorship: NIH-R15AI130988 Jin, Xing An, Sungjun Kightlinger, Weston Zhou, Jiacheng Hong, Seok Hoon 2021 Article islandora:1012026 http://hdl.handle.net/10560/islandora:1012026 ChBE / Chemical and Biological Engineering Illinois Institute of Technology en Creative Commons Attribution (CC BY) http://creativecommons.org/licenses/by/4.0/ Open Access