Project description:Analysis of changes in gene expression in Enterococcus faecalis OG1 delta-EF2638 mutant compared to wild-type OG1 strain. The deletion mutant has a growth defect when grown with aeration The mutant presented in this study is described and characterized in Vesic, D. and Kristich, C.J. 2012. A Rex-family transcriptional repressor influnces H2O2 accumulation by Enterococcus faecalis. (submitted for publication) Microarray analysis was done using RNA isolated from two independent cultures of wild-type Enterococcus faecalis OG1 and two independent cultres of Enterococcus faecalis OG1 delta-EF2638 mutant; each RNA sample was subjected to triplicate hybridization (technical replicates) . Microarrays were custom designed to investigate expression of ORFs in Enterococcus faecalis OG1RF genome. The arrays were designed based on the OG1RF annotation generated with the Rapid Annotation Using Subsystem Technology (RAST) server (Aziz et. al. 2008. BMC Genomics 9:75), as described in Frank et al (2012) Infect. Immun. 80:539. The aim was eighteen probe pairs per ORF, each of which is present in triplicate.
Project description:Detection of species-specific proteotypic peptides for accurate and easy characterization of infectious non-tuberculous mycobacteria such as Mycobacterium avium subsp. paratuberculosis is essential. Therefore, we conducted an in-depth global proteomic experiment using M. avium subsp. paratuberculosis ATCC 19698 (Map) strain followed by proteome database search and spectral library generation. The lysate was subjected to in-solution proteomic sample preparation and fractionated using an offline C18 StageTip. Each fraction was acquired in technical triplicates using a 180 min data-dependent acquisition (DDA) method in Orbitrap Fusion Tribrid (Thermo Scientific) mass spectrometer. The resulting raw DDA data were searched against the M. avium subsp. paratuberculosis proteome database using Proteome Discoverer and FragPipe. The resulting peptide spectrum matches were converted into a spectral library using BiblioSpec.
