ABSTRACT Chlamydia trachomatis represents a significant health concern world-wide. A substantial burden exists due to the prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular parasites that undergo a complex developmental cycle, and this biology has historically impeded rapid progress in understanding pathogenesis. Recently, the ability to transform Chlamydia with exogenous DNA has greatly facilitated advances in understanding the molecular mechanisms underpinning the success of chlamydial infection. For example, ectopic expression of chlamydial genes has provided insight into intracellular localization and function. As studies become more sophisticated, however, current promoter systems are often proving inadequate to address physiologically relevant biology. Progress is particularly hindered by limited choices in tightly- regulated, inducible promoters. We propose to address deficiencies by developing reagents for more finely tuned gene expression in Chlamydia. We will exploit advances in synthetic biology and rational design to engineer a suite of tightly regulated and tunable promoters. Preliminary data showing the efficacy of the approach are presented herein. We propose to validate and further develop these technologies. At the end of these studies, we will have established new approaches that will benefit the entire Chlamydia research community and advance the utility of ectopic gene expression to study chlamydial infection biology.