An Experimental Pipeline for Developing and Optimizing Genetic Tools in Genetic Intractable Gut Bacteria

The gut microbiota plays a crucial role in human health and disease, shaping both physiological and pathological outcomes in the host. Despite their significance, understanding the genetic underpinnings of their roles is challenging largely due to the lack of robust genetic tools for manipulating these organisms. We unveil an experimental pipeline tailored to craft genetic toolkits for manipulating gut bacteria traditionally deemed resistant to genetic manipulation. Our pipeline utilizes a conjugation-based method and offers detailed guidance to optimize (1) the genetic elements of the conjugative plasmid; (2) the conjugation procedures; and (3) the criteria for transconjugant selection, streamlining the manipulation process for increased simplicity and efficiency. As proof-of-principle for our approach, we applied it to two prominent and highly associated with human health, yet previously genetically intractable gut bacteria, Akkermansia muciniphila and Prevotella copri. We successfully engineered these microbes for both targeted (plasmid integration) and untargeted (transposon mutagenesis) genetic frameworks. This advancement enables us to (1) refine genetic tools for non-model bacteria, propelling functional genetic investigations in these organisms; (2) render these bacteria amenable for synthetic biology applications, enabling the creation of circuits, sensors, and switches that pave the way for innovative synthetic biology therapeutic interventions capturing ecological and taxonomic diversity in the gut.