Inducible Protein Knockdown via Engineered Small RNAs in Chlamydia

Genetic manipulation of Chlamydia trachomatis is challenging because of its obligate intracellular lifestyle and reduced genome with a high proportion of essential genes. Functional analysis of chlamydial genes often requires alternative genetic strategies such as conditional knockdown rather than gene disruption. This chapter describes the use of an engineered small RNA (sRNA) to achieve inducible protein knockdown in C. trachomatis. This approach utilizes the C. trachomatis sRNA CtrR3 as a scaffold to express a targeting sequence that interferes with a target mRNA by binding its 5' untranslated region (UTR), in the region of the ribosome-binding site (RBS) and start codon. The protocol detailed here covers design of the targeting sequence, bioinformatic analysis to minimize off-target binding, construction of the knockdown sRNA plasmid, generation of a chlamydial transformant expressing the engineered sRNA, induction of protein depletion, assessment of knockdown efficiency and phenotypes, and complementation to demonstrate knockdown specificity.