Tn7 Integration Efficiently Introduces Gene Expression Cassettes into a Gut-Native Escherichia coli Strain

Native bacteria are strains isolated from the host and often require greater development and optimization of genetic manipulation tools than lab-adapted strains. We have previously shown that the native murine Escherichia coli strain EcAZ-1, which colonizes the conventional mouse gut long term, is genetically tractable, and the engineered strain can elicit physiological effects while residing in the mouse gut. Given the interest in using EcAZ-1 and other native E. coli as a chassis for heterologous gene expression in the mouse gut, researchers need more efficient tools to rapidly and reliably introduce heterologous gene expression into the strain. Here, we demonstrate that a gene expression cassette can be integrated into the EcAZ-1 genome with high efficiency using the site-specific Tn7 transposon. Furthermore, we demonstrate that EcAZ-1 modified to express sfGFP using this method stably engrafts in the gut of conventional mouse. Taken together, Tn7-mediated integration is an effective method for stably introducing genetic cassettes into the native murine strain EcAZ-1 for expression in the mouse gut.