Browsing by Author "University of Minnesota Tischler Lab"

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    Mycobacterium tuberculosis Erdman ATCC 35801 arrayed Himar1 transposon library isolated using MtbYM rich medium.
    (2022-08-18) Tischler, Anna D; Block, Alisha; Palani, Nagendra; Brokaw, Alyssa; Zhang, Leanne; Namugenyi, Sarah; Beckman, Kenneth; tischler@umn.edu; Tischler, Anna D; University of Minnesota Tischler Lab
    We created a Himar1 transposon library in Mycobacterium tuberculosis Erdman ATCC 35801 strain containing ~8000 mutants arrayed in 80 tube racks in a 96-well format. The transposon library was isolated on MtbYM rich medium that allowed recovery of auxotrophic mutants that would not be viable on standard Middlebrook M. tuberculosis culture medium. Using orthogonal pooling and transposon sequencing (Tn-seq), we mapped the location of Tn mutants in the library. The library was split into two groups of 40 racks. For each set of 40 racks, pools were created of all mutants in each row (rows A-H), column (columns 1-12) and rack (racks 1-40 or 41-80) to generate 60 Tn-seq samples for Illumina sequencing. Two different mapping methods, the heuristic Straight Three strategy and the probabilistic Knockout Sudoku algorithm, were used to map the arrayed library with good agreement between both mapping methods. This repository contains Illumina sequencing data for all rack, column, and row pools used to map the entire 80 rack arrayed library.
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    Tnseq screen of Mycobacterium tuberculosis Himar1 transposon library grown in MtbYM rich medium and treated with antitubercular drugs.
    (2022-08-15) Block, Alisha M; Tischler, Anna D; tischler@umn.edu; Tischler, Anna D; University of Minnesota Tischler Lab
    Transposon-sequencing (Tn-seq) is a powerful genetic tool that can identify changes in transposon (Tn) mutant frequency during a given test condition such as drug treatment during nutrient starvation. We created an arrayed library of Tn mutants in Mycobacterium tuberculosis Erdman, screened a subset of the library (~1,000 mutants) by Tn-seq, and identified 100+ Tn mutants with altered tolerance to antibiotics in starvation conditions. We individually retested three Tn mutants to confirm their Tn-seq phenotypes, rv0457c::Tn, ppgK::Tn and clpS::Tn, and all three mutants showed reproducible increased susceptibility to drug treatment during starvation. However, the phenotypes for two of Tn mutants, ppgK::Tn and clpS::Tn, were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed that both Tn mutants had single nucleotide polymorphisms that prevented PDIM production, which was responsible for the observed drug tolerance phenotypes. This repository contains all the Illumina sequencing data for the M. tuberculosis antibiotic tolerance Tn-seq experiments and whole-genome sequencing data for our lab WT Erdman strain and three Tn mutants identified in the Tn-seq screen that were individually retested.