A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in C. trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid ba..., These data represent source data from the manuscript entitiled: A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis
The raw data are qPCR, immunoblots, immunofluorescent images,and  DNA agarose gels. 
C. trachomatis serovar L2 (LGV 434) was used as the parent strain in these studies. Chlamydiae was maintained in HeLa 229 epithelial cells (CCL-1.2; ATCC) for routine culture and for genetic manipulations. HeLa were grown in RPMI 1640 medium containing 2 mM L-glutamine (Life Technologies) supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS; Sigma).  All cultures were maintained at 37 °C in an environment with 5 % CO2 and 95 % humidified air. Infections were accomplished by centrifugation of EBs onto cell monolayers at 20 °C for 1h at 900 x g. Where appropriate, cultures were supplemented with 2 µg/ml cycloheximide (Cyclo), 50 ng/ml anhydrotetracycline (aTc), and/or 2 mM Theophylline (Theo; Sigma).nd chlamydiae.
Plasmid ..., , # A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

[https://doi.org/10.5061/dryad.f1vhhmh6d](https://doi.org/10.5061/dryad.f1vhhmh6d)

#### Description of the data and file structure

These data were collected as part of a study to develop an efficient transposon mutagenesis system for C. trachomatis. The data were collected from HeLa cultures infected with C. trachomatis expressing pOri-Tn(Q). For qRT-PCR and qPCR analyses DNA was extracted from triplicate infected cultures and processed for respective amplification in triplicate. Raw CT values are shown for all reactions.  For immunoblot analyses, whole culture samples were concentrated by TCA precipitation and resolved in SDS-PAGE gels. Immunoblots were probed with Chlamydia-specific or transposase-specific (C9). antibodies. For immunofluorescence, infected cultures were fixed and probed with Chlamydia-specific antibodies (indirect) or visualized for mCherry fluorescence (direct)....