Analysis of Chlamydia trachomatis Gene Function via Controlled Up- or Downregulation

Due to genome streamlining, many genes in the ~1 Mb genome of Chlamydia trachomatis are essential and cannot be deleted. Ectopic overexpression and CRISPRi-based knockdown are complementary genetic approaches to study the function of genes in C. trachomatis, including essential genes. When used in combination with inducible promoters, these tools allow researchers to determine how precise regulation of gene expression contributes to proper progression of the chlamydial developmental cycle. This chapter includes protocols which detail ectopic overexpression of a protein or conversely, transcriptional inhibition of a specific gene in C. trachomatis serovar L2/434/Bu.