Jin, Xing Creator An, Sungjun Creator Kightlinger, Weston Creator Zhou, Jiacheng Creator Hong, Seok Hoon Creator Faculty shong26@iit.edu https://orcid.org/0000-0003-2401-6368 ChBE / Chemical and Biological Engineering Illinois Institute of Technology Affiliated department Biological Engineering Biotechnology 2021 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. NIH-R15AI130988 Article en https://doi.org/10.1002/aic.17466 Creative Commons Attribution (CC BY) http://creativecommons.org/licenses/by/4.0/ Open Access eng http://hdl.handle.net/10560/islandora:1012026