Construction of Inducible Transposon Mutant Libraries in Chlamydia trachomatis.

Understanding the minimal gene set required for cellular viability is a central goal in microbiology. A transposon mutagenesis protocol has been developed for use in Chlamydia to enable gene function analysis and reveal the essential genome. This method utilizes a self-replicating Himar-1 transposon delivery system, with a hyperactive transposase gene under inducible transcriptional and translational control, coupled with long-read nanopore sequencing to identify transposon insertion sites. In a step-by-step protocol, we describe how to prepare the transposon library by infecting cells with a Chlamydia strain pre-transformed with the transposon delivery vector and inducing expression of the transposase. After selection for survival, the library DNA is sequenced by long-read nanopore sequencing and analyzed with bioinformatic tools to identify transposon-genome junctions. This strategy generates ~30 unique insertions per experiment in the C. trachomatis input library.