Escherichia coli
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA288458 | ENA |
REPOSITORIES: ENA
Dataset's files
primary
| Action | DRS | |||
|---|---|---|---|---|
| SRR2080776.fastq.gz | Fastqsanger.gz |
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Project description:Study of the mechanisms of RecB mutant terminus DNA loss in Escherichia coli. FX158: WT MG1655 FX35: recB- FX37: ruvAB- FX51: matP- MIC18: recB- sbcD- sbcC- MIC20: recB- ruvAB- MIC24: matP- recB- MIC25: recA- recB- MIC31: sbcB- sbcD- MIC34: recA- recD- MIC40: linear chromosome MIC41: linear chromosome recB- MIC42: matP- ftsKC- MIC43: matP- ftsKC- recB- MIC48: recA- Cells were grown in M9 minimal medium supplemented with 0.4 % glucose to exponential phase (0.2 OD 650 nm). Chromosomal DNA was extracted using the Sigma GenElute bacterial genomic DNA kit. 5 μg of DNA were used to generate a genomic library according to Illumina's protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (http://www.i2bc.paris-saclay.fr/spip.php?article399&lang=en, CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations. Library quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent technologies). Libraries were pooled in equimolar proportions. 75 bp single reads were generated on an Illumina MiSeq instrument, using a MiSeq Reagent kit V2 (500 cycles) (Illumina), with an expected depth of 217X. An in-lab written MATLAB-based script was used to perform marker frequency analysis. Reads were aligned on the Escherichia coli K12 MG1655 genome using BWA software. Data were normalized by dividing uniquely mapping sequence reads by the total number of reads. Enrichment of uniquely mapping sequence reads in 1 kb non-overlapping windows were calculated and plotted against the chromosomal coordinates.
2017-11-10 | E-MTAB-6122 | ArrayExpress
Project description:Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCE Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.
2018-01-01 | S-EPMC5821084 | BioStudies
Project description:BACKGROUND: A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates. FINDINGS: The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions. CONCLUSIONS: The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.
2011-01-01 | S-EPMC3146853 | BioStudies
Project description:Chemical genomics expands our understanding of microbial tolerance to inhibitory chemicals, but its scope is often limited by the throughput of genome-scale library construction and genotype-phenotype mapping. Here we report a method for rapid, parallel, and deep characterization of the response to antibiotics in Escherichia coli using a barcoded genome-scale library, next-generation sequencing, and streamlined bioinformatics software. The method provides quantitative growth data (over 200,000 measurements) and identifies contributing antimicrobial resistance and susceptibility alleles. Using multivariate analysis, we also find that subtle differences in the population responses resonate across multiple levels of functional hierarchy. Finally, we use machine learning to identify a unique allelic and proteomic fingerprint for each antibiotic. The method can be broadly applied to tolerance for any chemical from toxic metabolites to next-generation biofuels and antibiotics.
2016-01-01 | S-EPMC4714920 | BioStudies
Project description:A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.
1000-01-01 | S-EPMC2980817 | BioStudies
Project description:Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-lacZ-aph-P(lac)-FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans. Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of yacF, yacG, yacH, yacK (cueO), yacL, ruvA, ruvB, yabB, and yabC made with the cassette were used to verify its properties.
2002-01-01 | S-EPMC135234 | BioStudies
Project description:The natural history of the amoeba Dictyostelium discoideum has inspired scientific inquiry for seventy-five years. A genetically tractable haploid eukaryote, D. discoideum appeals as a laboratory model as well. However, certain rote molecular genetic tasks, such as PCR and cloning, are difficult due to the AT-richness and low complexity of its genome. Here we report on the construction of a ~20 fold coverage D. discoideum genomic library in Escherichia coli, cloning 4-10 kilobase partial restriction fragments into a linear vector. End-sequencing indicates that most clones map to the six chromosomes in an unbiased distribution. Over 70% of these clones contain at least one complete open reading frame. We demonstrate that individual clones and library composition are stable over multiple replication cycles. Our library will enable numerous molecular biological applications and the completion of additional species' genome sequences, and suggests a path towards the long-elusive goal of genetic complementation.
2015-01-01 | S-EPMC4575855 | BioStudies
Project description:A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2. To discuss this variation, we adopt the term 'framework' to refer to genes that are stable components of genomes within related lineages, while 'migratory' genes are transient inhabitants of the genome. Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined. The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation. The patterns observed are consistent with the action of a site-specific recombinase. Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked.
2004-01-01 | S-EPMC373321 | BioStudies
Project description:In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.
1000-01-01 | S-EPMC523700 | BioStudies
Project description:Flagella contribute to the virulence of bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellar phase variation is believed to facilitate bacterial evasion of the host immune response. In this study, the flnA gene that encodes Escherichia coli H17 flagellin was examined by whole genome sequencing and genetic deletion analysis. Unilateral flagellar phase variation has been reported in E. coli H3, H47 and H17 strains, although the mechanism for phase variation in the H17 strain has not been previously understood. Analysis of phase variants indicated that the flagellar phase variation in the H17 strain was caused by the deletion of an ∼35 kb DNA region containing the flnA gene from diverse excision sites. The presence of covalently closed extrachromosomal circular forms of this excised 35 kb region was confirmed by the two-step polymerase chain reaction. The deletion and complementation test revealed that the Int1157 integrase, a tyrosine recombinase, mediates the excision of this region. Unlike most tyrosine recombinases, Int1157 is suggested to recognize diverse sites and mediate recombination between non-homologous DNA sequences. This is the first report of non-homologous recombination mediating flagellar phase variation.
2012-01-01 | S-EPMC3378880 | BioStudies