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The emergence of antibiotic resistant bacteria has been a global concern for numerous decades. Bacteria are able to attain resistance to many current antibiotics due to high mutation rates and the ability to share genomic DNA with other bacterial species. New pathways need to be exploited for drug design studies, not only to limit the spread of resistance but also to generate a greater diversity in antimicrobial mechanisms. Tryptophan is an essential amino acid that is synthesized from enzymes found only in plants and bacteria. The lack of any enzyme homologues for humans makes this pathway an ideal target for inhibition studies. We have previously shown in laboratory studies that bacterial mutants lacking the tryptophan gene are unable to grow in tryptophan deprived media, further demonstrating the potential for drug design studies against it. E. coli Tryptophan synthase (TrpS), the enzyme catalyzing the last two steps in the biosynthetic pathway of tryptophan, was chosen for inhibitor compound screening studies. TrpS consists of two distinct subunits (TrpS-? and TrpS-?), each able to catalyze their own reaction. TrpS-? and TrpS-? protein were separately cloned, expressed and purified to >90% purity using recombinant-expression techniques. Assays were developed for the TrpS-? and TrpS-? enzymes to assess their catalytic activity and the TrpS-? assay was converted to a 96-well plate format for initial inhibitor screening. In vitro and in silico screens using a first generation library of compounds generated against IGPS, the enzyme just prior to TrpS in the tryptophan biosynthetic pathway, resulted in several potential inhibitors for future lead compound screening development. The results of this study demonstrate the potential to develop a new class of inhibitors against E. coli TrpS.