ArticleMethods in molecular biology (Clifton, N.J.)2027
Construction of Inducible Transposon Mutant Libraries in Chlamydia trachomatis.
Article in Methods in molecular biology (Clifton, N.J.), 2027. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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