The human large intestine houses trillions of microorganisms which collectively form the highly diverse microbial community known as the gut microbiota. The gut microbiota performs many functions critical to the maintenance of health, including extraction of nutrients from food, production of vitamins, and defense against pathogens. Like an organ, disturbances to the structure of the gut microbiota can have significant negative impacts to the human host, including obesity, malnutrition, and cancer. However, the gut microbiota is currently unique among organs in that it is highly engineerable, enabling improvement of function by substituting beneficial microbes for less desirable ones. These exogenous beneficial microbes are termed “probiotics” and their close contact with both their human host, as well as other gut bacteria, raises exciting therapeutic prospects, including the provision of additional metabolic functions, modulation of the host immune response, or competitive exclusion of pathogens. However, these applications remain out of reach due to the low residence time of most probiotics within the gut, as well as an inability to reliably control gene expression in probiotics residing in the gut environment. In this talk, I will show exciting data from a culture-independent bioprospecting approach for increasing the residence time and altering the biogeography of probiotic strains within the gut. Then, I will discuss the optimization and application of a high-throughput sequencing-based approach to the parallel characterization of many transcriptional regulators in gut-resident probiotics. Taken together, this work has the potential to significantly improve our understanding and use of probiotics while providing a framework for developing robust chassis strains for future synthetic biology efforts in the human gut microbiota.