Project description:Protein expression by E. coli 26561 during the late-exponential phase of cultures under anaerobic conditions was examined. E. coli 26561 is a multidrug resistant (MDR) and shows an unusual hyper-mucoviscous phenotype. Resistance includes ESBL (CTX-M-14) and proteome was determined with and without exposure to sub-MIC concentrations of the 3rd generation cephalosporin ceftazidime. Ceftazidime exposure was at two sub-MIC levels, specifically 0.25x MIC (samples 5-7), 0.5x MIC (samples 8 - 10); samples 1-4 provided the unexposed Control. Both whole and phospho-enriched fractions for each sample were analysed. Quantification of peptides was assessed using 10-plex TMT labelling in conjunction with an Orbitrap Fusion Tribrid. Raw data produced by the Orbitrap were processed using Max Quant 1.5.4.7 using the included Andromeda search engine. Peptides were searched against our own database of E. coli 26561 proteins which was produced from a hybrid assembly of our reads obtained from MiSeq and PacBio sequencing platforms.
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.
Project description:Purpose: In this study, Escherichia coli DH5alpha whole transcriptome sequencing was performed in order to compare the different gene expression profiles between control and exposed to Wi-Fi radiofrequency radiations. Methods:Escherichia coli DH5alpha were exposed to Wi-Fi radiations. Total RNA samples( control and exposed ) were extracted by bacteria protect-Rneasy kit,treated with DNAase and subjected to sequnecing using an Illumina-NovaSeq 6000 platform. Library preparation and sequencing were performed by Macrogen (south korea).Trimmed reads are mapped to reference genome with Bowtie. HTseq was used for expression profiling. Expression profile was calculated for each sample and gene as read count.
Project description:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Keywords: addition of quorum and host hormone signals
Project description:We performed a high-throughput mapping of the 5’ end transcriptome of the pAA plasmid of the clinical Escherichia coli O104:H4 (E. coli O104:H4) isolate LB226692. We employed differential RNA-sequencing (dRNA-seq), a terminator exonuclease (TEX)-based RNA-seq approach allowing for the discrimination of primary and processed transcripts. This method has proven to be a powerful tool for the mapping of transcription start sites (TSS) and detection of non-coding RNAs (ncRNAs) in bacteria. We catalogued pAA-associated TSS and processing sites on a plasmid-wide scale and performed a detailed analysis of the primary transcriptome focusing on pAA virulence gene expression.
Project description:In this study transcriptional start sites (TSS) of E. coli were determined for several growth conditions To detect the complement of transcripts expressed from E. coli, we collected two independent biological replicates (B1 and B2 samples) from MG1655 wild type strain grown to exponential (OD 600 ~0.4) or stationary phase (OD 600 ~2.0) in LB medium (samples LB 0.4 and LB 2.0, respectively) as well as grown to exponential phase (OD 600 ~0.4) in M63 minimal glucose medium (sample M63 0.4). For all six samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5M-bM-^@M-^Y terminator exonuclease (+TEX samples), which degrades RNAs containing a 5M-bM-^@M-^Y-mono-phosphate (5M-bM-^@M-^Y-P) and, thus, enriching enriches for primary transcripts containing 5M-bM-^@M-^Y-tri-phosphates (5M-bM-^@M-^Y-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5M-bM-^@M-^Y-PPP) and processed RNAs (5M-bM-^@M-^Y-P).
Project description:Sequencing reads of Escherichia coli K-12 control and antibiotic-treated cultures enriched (Cappable-Seq) and not enriched (Ultra directional) for the 5’ end of primary transcripts.
Project description:Counting DNA reads using whole genome sequencing is providing new insight into DNA double-strand break repair (DSBR) in the model organism Escherichia coli. We describe the application of RecA chromatin immunoprecipitation coupled to genomic DNA sequencing (RecA-ChIP-seq) and marker frequency analysis (MFA) to analyse the genomic consequences of DSBR.
Project description:Comparison of the whole genome gene expression level of an amoxicillin resistant E. coli strain with the wildtype it was derived from. The process of amoxicillin adaptation of E. coli MG1655 wildtype cells is further descibed in van der Horst, M, J.M. Schuurmans, M. C. Smid, B. B. Koenders, and B. H. ter Kuile (2011) in Microb. Drug Resist. 17:141-147. Resistance to amoxicillin was induced in E. coli by growth in the presence of stepwise increasing antibiotic concentrations. To investigate consequences of the aquisition of amoxicillin resistance the transcriptomic profile of sensitive and resistant cells was compared in the absence and presence of sub-inhibitory (0.25xMIC) amoxicillin concentrations was compared.
Project description:In order to understand the impact of genetic variants on transcription and ultimately in changes in observed phenotypes we have measured transcript levels in an Escherichia coli strains collection, for which genetic and phenotypic data has also been measured.