Abstract
As the understanding of bacteria-mediated cancer therapies deepens, bacteria such as Escherichia coli Nissle 1917 (EcN) have become a promising platform for cancer therapy. However, their potential role in real-time monitoring and visualization tools still needs to be explored and enhanced. In this study, we aimed to screen and optimize EcN visualization systems for non-invasive in vivo bioluminescence imaging in live mice. To this end, we developed three series of recombinant EcN strains expressing Gaussia luciferase (Gluc), Renilla luciferase (Rluc), and NanoLuc (Nluc), along with their respective mutants. These strains exhibited bioluminescence when different coelenterazine (CTZ) substrates were present. As a result, multiple bioluminescent EcN strain-substrate pairs were identified with stronger, longer, or red-shifted bioluminescence, offering multiple effective optical tumor-targeting systems for in vivo studies investigating bacteria-mediated cancer therapy and intestinal diseases.