When introduced to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid, an exopolysaccharide that completely engulfs the organism, to establish a protective barrier between the organism and the acidic environment. The colanic acid polymer is made up of multiple six-sugar repeating units comprised of glucose, 2,3-acetylated fucose, fucose, galactose, glucoronic acid and 4,6-pyruvated galactose. Studies have shown that as pH of an environment is lowered, E. coli strains excreting colanic acid survive longer than strains that cannot produce the polymer. This action may contribute to the survival of virulent E. coli in contaminated food products and inside a host organism. Considering the contaminated food sources containing these bacteria are widespread and contribute to over 73,000 infections per year in the United States alone, it is vitally important to understand how colanic acid is synthesized to develop novel antibiotic therapeutics to target this pathway. While the region of the E. coli genome that encodes for colanic acid biosynthesis has been reported, only the first enzyme in the biosynthesis pathway has been characterized. Utilizing fluorescent analogues to bactoprenyl monophosphate, the isoprenoid anchor that colanic acid is built upon, the next three glycosyltransferase enzymes (WcaI, WcaE, WcaC) in the pathway have been characterized. Finally, the data presented here suggests that the acetylation of the first fucose residue is regulatory in colanic acid biosynthesis and therefore could be a potential antibiotic target for this pathway.