Ontology highlight
ABSTRACT:
PROVIDER: PRJNA422683 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6382500.fastq.gz | Fastqsanger.gz | |||
| SRR6382510.fastq.gz | Fastqsanger.gz | |||
| SRR6382491.fastq.gz | Fastqsanger.gz | |||
| SRR6382501.fastq.gz | Fastqsanger.gz | |||
| SRR6382511.fastq.gz | Fastqsanger.gz |
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Project description:The transposon site hybridization (TraSH) technique (Sassetti, CM et al. 2001. PNAS 98:12712-7) was utilized to identify genes important for the survival of Y. pestis within murine macrophages. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool) in order to identify Y. pestis genes important for survival in macrophages. Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs can be used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool). Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs was used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe.
2011-11-30 | E-GEOD-33982 | biostudies-arrayexpress
Project description:The transposon site hybridization (TraSH) technique (Sassetti, CM et al. 2001. PNAS 98:12712-7) was utilized to identify genes important for the survival of Y. pestis within murine macrophages. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool) in order to identify Y. pestis genes important for survival in macrophages. Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs can be used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe.
2011-11-30 | GSE33982 | GEO
Project description:Acetobacter fabarum DSM 19596 genome sequencing project
| PRJDB1013 | ENA
Project description:A Tn5 transposon insertion library generated in a thyA- strain was selected in thymidine-free media and insertions were mapped and sequenced at different time points to look for changes in survival caused by gene disruptions
2022-10-11 | GSE147760 | GEO
Project description:Mapping of transposon mutant library during growth in Brain Heart Infusion (BHI) broth and in a rat endocarditis model. The goal of this study was to identify factors that play a role in E. faecium endocarditis by selection of transposon insertion mutants that lost the capacity to cause infections.
2016-03-24 | E-MTAB-4523 | biostudies-arrayexpress
Project description:Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae. Keywords: GAF Identification of transposon insertion sites
2007-06-26 | GSE6913 | GEO
Project description:Mapping of transposon mutant library in Enterococcus faecium during growth in Brain Heart Infusion (BHI) broth and in a semi-static biofilm model. The goal of this study was to identify factors that play a role in E. faecium biofilm formation by selection of transposon insertion mutants that lost the capacity to form biofilm in vitro.
2016-05-01 | E-MTAB-4524 | biostudies-arrayexpress
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.
2024-07-06 | E-MTAB-11576 | biostudies-arrayexpress
Project description:The aim of the experiment was to assay every gene in the E. coli genome to identify those that contribute to increased or decreased susceptibility to the beta-lactam antibiotic meropenem. A library of transposon-insertion mutants was grown overnight in the presence or absence of a range of concentrations of meropenem. Different concentrations of IPTG were also used to promote expression from a transposon-encoded promoter to assay the effects of increased transcription for each gene. DNA sequencing was then used to reveal the locations of the transposons in every mutant. By comparing the numbers of each mutant between conditions, information can be gained about the relative fitness of that mutant under the conditions tested.
2022-06-09 | E-MTAB-11802 | biostudies-arrayexpress