The microbial community resident in the human gut provides a variety of functions critical to host health and well-being. The vast majority of gut microbes remain uncultured and much of our insight and appreciation of the functional capacity of the microbiota has been provided by culture independent metagenomic sequencing. Indeed, the establishment of the “human microbiome” as a distinct field of research has been underpinned by rapid technological advances in DNA sequencing methods and computational analyses. This has provided an unprecedented insight into the genetic potential of the microbiota and transformed our awareness and appreciation of the extent of microbial “dark matter” that exists in these communities. However, recent analyses have revealed that the coverage of gene diversity is increasingly saturated although the overwhelming majority of gene products remain functionally uncharacterised. It is indisputable that genetic techniques have been instrumental over many decades in linking genes to function and elucidating the functional capacity of the microbial world. The key constraints to characterising the gut microbiota are that the majority of isolates are not known to be genetically tractable and there is a paucity of genetic tools with which to rationally characterise these microbes. We recently described a modular vector system and an innovative approach termed “metaparental mating” that supports the rapid and directed isolation of genetically tractable fastidious gut bacteria. We applied the vector system to support the isolation of genetically competent human colonic Firmicutes as these bacteria are functionally diverse but significantly underrepresented in existing bacterial culture collections. We have now extended the functionality of the vector system to support the dissection of fastidious gut microbes by forward and reverse genetic means. Collectively, this offers new opportunities to link genes with function and may support the development of novel biotechnologies and therapeutics that improve gut function. Critically, this approach could be applied to facilitate the functional characterisation of other microbial ecosystems.