Biofilms can be defined as a complex aggregation of bacterial communities that involves many gene regulatory mechanisms, as well as evolutionary processes to increase biodiversity. Specific Aim 1 used a gene regulation approach to identity novel targets for the development of biofilm prevention and treatment techniques. The goal was to determine genes that get expressed early in biofilm development (prevention targets) and genes that get expressed late and in the outer layer of the biofilm (treatment targets). Biofilm formation is regulated by numerous regulators, including the two-component osmoregulator system EnvZ/OmpR, the colanic acid activator rcsCDB and the global regulator FlhD/FlhC. In this study, we determined the temporal and spatial expression of flhD, ompR and rcsB in E. coli k-12 AJW678 biofilm, as well as the gene expression of flhD in isogenic ompR and rcsB mutants. Results indicated that flhD was expressed early, and in the outer layer of the mature biofilm. We concluded that FlhD/FlhC would be the first target for novel prevention and treatment technique. One mechanism to increase biodiversity in biofilm is the insertion of transposon elements, which was investigated as Specific Aim 2. Insertion of IS elements into the flhD promoter resulted in increased motility in numerous E. coli K-12 strains has been shown in previous study. In this study, we recovered isolates from biofilm, where IS1 had inserted in the flhD promoter further downstream than in previously described strains. These isolates showed reduced motility. We also wanted to determine the effect of an IS element insertion on regulation of flhD expression by OmpR and RcsB in biofilm. Temporal and spatial gene expression of three different GFP-tagged flhD promoters was measured. The results indicated that IS5 insertion in the flhD promoter at the published hotspot did not have any effect on regulation of flhD expression by OmpR and RcsB in biofilm.