Most bacteria live in surface-attached communities called biofilms, where they play profound roles in many important processes in infection, industry, agriculture, and the natural environment. A number of different genetic pathways have been demonstrated to impact biofilm formation at the aggregate level, typically by showing that the amount of biofilm produced by mutants lacking these pathways is either reduced or abolished altogether. However, we know very little about the how these pathways contribute to biofilm architecture at the scale of single cells, nor how these pathways interact with one another during biofilm development. This is because biofilms are three-dimensional, translucent structures, which makes it difficult to visualize what is happening deep inside of them. This project will use transformative new approaches to understand how bacteria work together to assemble biofilms. We hypothesize that multicellular biofilms undergo a series of developmental processes analogous to those observed in animals, where different pathways exhibit complex patterns of gene expression. These processes allow a single cell to transform into a multicellular biofilm, where genetically identical cells perform different roles and functions. Because we cannot currently visualize cells deep inside a mature biofilm, we will use microfluidic devices to approximate a two-dimensional cross-section through a mature biofilm, allowing us to directly visualize the behavior of individual cells and measure the expression of multiple genetic pathways simultaneously over long time periods. We will then use cell tracking and mutants to understand how each of these pathways control the behavior of individual cells within the 2D biofilm. These results will be validated in 3D biofilms, using separate microfluidic devices formed with fluid walls, which will allow us to extract fully intact biofilms for subsequent analysis by novel electron microscopy assays and by single-cell transcriptomics. By quantifying how each pathway contributes to biofilm assembly, and how each of these pathways interact with one another, we will provide an unprecedented view of the sequence of processes that play out in space and time during biofilm development. Such insights hold the key for developing new strategies for both rationally engineering beneficial biofilms and eradicating harmful biofilms.