Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Whole genome \"TraDIS-Xpress\" assay to determine gene roles during growth of E. coli in different concentrations of the antibiotics trimethoprim and sulfamethoxazole

ABSTRACT: To assay every gene in the E. coli genome to identify those that contribute to increased or decreased susceptibility to the antibiotics trimethoprim and sulfamethoxazole. This will help to define more accurately those bacterial cell mechanisms that contribute to these phenomena and provide information that will contribute to the development of new antibiotics, or compounds or known antibiotics that synergise with those already in clinical use. Thus, this set of experiments confirmed that AZT, widely known for its antiviral activity, acts synergistically with trimehoprim.

INSTRUMENT(S): NextSeq 550

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Arthur Turner

PROVIDER: E-MTAB-9305 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Yasir Muhammad M Turner A Keith AK Bastkowski Sarah S Baker David D Page Andrew J AJ Telatin Andrea A Phan Minh-Duy MD Monahan Leigh L Savva George M GM Darling Aaron A Webber Mark A MA Charles Ian G IG

Genome research 20200212 2

Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an in ...[more]

PMID: 32051187

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Project description:A whole genome screen was used to assay every gene of Escherichia coli strain BW25113 to identify genes involved in susceptibility to the monobactam (beta-lactam) antibiotic aztreonam. The methodology has been called TraDIS-Xpress, and is a version of TraDIS or Tn-seq. A transposon mutant library consisting of several hundred thousand mutants was constructed using a Tn5-derived transposon incorporating an inducible outward transcribing promoter. All the mutants were grown in LB broth cultures supplemented with aztreonam at 2 x, 1 x, 0.5 x and 0.25 x MIC with induction of the transposon promoter using 0.2 mM IPTG or 1 mM IPTG or without induction. Following growth, mutants with increased susceptibility show reduced numbers and those with reduced susceptibility show increased numbers. Each condition was performed in duplicate. The methodology enable genes to be assayed by insertional inactivation or by changes in expression. Expression changes result from altered transcription from upstream transposon insertions transcribing into the gene, or downstream insertions transcribing into the gene in the reverse direction leading to RNA interference through the generation of reverse and complementary RNA. Thus, essential genes into which transposon insertions are not tolerated may be assayed also by changes in numbers of upstream or downstream insertion mutants. Changes to high throughput sequencing protocols permit the generation of nucleotide sequence reads from the known transposon sequences into the surrounding insertion site for all the mutants in the mixture simultaneously. Matching the sequence of the reads to the genome nucleotide sequence of E. coli BW25113 then allows the precise locations of all the transposon insertion sites of all the mutants to be mapped simultaneously. The relative changes in mutants between control (without) and selective condition (with aztreonam) then indicates which genes are involved in susceptibility. The numbers of sequence reads that match is reflected by the number of mutants, and so the degree of susceptibility can also be estimated.

Dataset Information

Whole genome \"TraDIS-Xpress\" assay to determine gene roles during growth of E. coli in different concentrations of the antibiotics trimethoprim and sulfamethoxazole

Publications

TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Similar Datasets

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