Bacteria are the most popular chassis for synthetic biology and industrial biotechnology, owing to the comparative ease of their genetic engineering toward desired functions. Synthetic biology has enabled this goal through systematic gene expression control, by abstracting gene regulatory elements as plug and play modules, including promoters, ribosome binding sites, protein-coding sequences, and terminators. These parts are not perfectly modular and frequently fail to function as predicted in a gene or circuit, owing to compositional effects, chassis environment, etc. Furthermore, the specific parts needed to meet a circuit’s performance criteria are often unknown. Rapid prototyping of DNA is necessary to eliminate bottlenecks in the design-build-test cycle of fast-growing bacteria, and physical standardization and sharing of genetic parts and modules across research labs is a promising solution. The Bacterial Toolkit hierarchical DNA assembly system developed in this thesis and the companion part collection allows complete specification of transcriptional and translational cassette elements in custom combinations of mono- and polycistronic operons in any orientation, to efficiently build bacterial genetic circuits, pathways, and vectors for diverse needs from physically interchangeable parts. A distribution of modern gene expression parts is also provided, including promoters, insulators, insulated ribosome binding sites, transcription factors, protein tags, strong terminators, common E. coli and broad-host range origins, selection markers, plasmid maintenance factors, and E. coli genomic integration homologies. The Bacterial Toolkit is already in use by researchers and has been used for the construction of numerous genetic circuits with functions spanning bacterial pattern formation and population control, asymmetric plasmid partitioning, RNA logic, gene expression library construction, glucose biosensing, bioremediation, protein biomaterials, and environmental monitoring of horizontal gene transfer. Here, we used the Toolkit to demonstrate high assembly fidelity and streamlined genomic integration, test transcriptional insulation afforded by connector elements, investigate the design strategies for UTR insulation in operons, and measure the effects of gene compositional contexts on promoter performance.