dc.title: Improved genetic tools and evolution-aware approaches for engineering honey bee gut symbionts dc.description.abstract: The Western honey bee (Apis mellifera) is an indispensable agricultural pollinator supporting a wide variety of food crops. Unfortunately, widespread use of pesticides, pathogens like deformed wing virus, and invasive pests including hive beetles, killer hornets, and Varroa mites, have threatened managed bee colonies and have led to record high rates of colony losses throughout the world. One promising approach to improve honey bee health is to engineer symbiotic gut bacteria to protect against these threats. Snodgrassella alvi is a robust, primary colonizer of the bee gut that can be readily grown in laboratory culture and naturally benefits host development, metabolism, and immunity. These characteristics make S. alvi an excellent candidate for symbiont-mediated applications. It has been genetically engineered to alter how it interacts with other bacteria in the bee gut community and with the bee host, to induce protective RNA interference responses against bee viruses and pests, and to sense a chemical signal in the bee diet. However, the question remains: how consistent, stable, and translatable are these tools? In this dissertation, I begin by providing a broad overview of honey bees, their gut microbiome community, and the need for consistent, stable, and translatable tools for engineering S. alvi (Chapter 1). Next, I describe new genetic engineering approaches that can be used to modify the S. alvi chromosome to decrease dependency on plasmids and reduce the use of antibiotic resistance genes. I also tested inducible promoter systems that can be used to activate expression of heterologous genes only when they are needed (Chapter 2). When we engineer a symbiont outside its native environment, we must pay attention to how it is evolving to those conditions and whether mutations that occur in the laboratory environment will compromise its ability to reestablish in the host. I found that S. alvi rapidly adapts to laboratory conditions and this can impair its ability to colonize and be transmitted between bees. To mitigate this issue, I propose ways current culture methods can be modified to prevent unwanted evolution (Chapter 3). While studying evolution of S. alvi in the bee gut, I serendipitously discovered that it is naturally competent. I investigated its DNA uptake mechanism, confirmed that competence is not limited to its native environment, and explored how we can harness this ability for engineering (Chapter 4). Finally, I conclude by discussing how these findings will improve the reliability of symbiont engineering approaches for studying and protecting bees and discuss future directions for expanding the synthetic biology toolkit for bee gut symbionts (Chapter 5).