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 126.96.36.199 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:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Keywords: addition of quorum and host hormone signals Overall design: These E. coli strains were grown with various signaling molecules and the expression profiles were determined using the Affymetrix E. coli 2.0 Array. Comparisons of the profiles were used to guide further examination using RT-PCR.