An Optimized Electrotransformation Protocol forLactobacillus jensenii

Abstract

ABSTRACTEngineered bacteria are promising candidates forin situdetection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol forLactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5·103CFUs/µg of DNA inL. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression inL. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates ofL. jensenii. This work will facilitate the genetic engineering ofL. jenseniiand enable its use for addressing challenges in gynecological healthcare.