Project description:Effect of Thiol reductive stress (DTT 1mM) on M.tb Rv was studied. DTT reduced at 1 mM concentration was added to log pahse culture of M.tb H37Rv. After Three hours RNA was isolated and hybridization was done on HRI-UMNDJ Mycobacterium tuberculosis 4.8K CAG_Mtb microarrays.
Project description:Expression profile of Mycobacterium tuberculosis H37Rv biofilm as induced by DTT (Reduced) 6mM DTT reduced at 6 mM concentration was added to log phase culture of Mtb H37Rv. After 29 hours RNA was isolated and hybridization was done on microarrays
Project description:In this study, RNA sequencing (Transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb infected macrophages and then sequenced.
Project description:In this study, we used GC-MS analysis in combination with flux analysis and the Affymetrix ATH1 GeneChip to survey the metabolome and transcriptome of Arabidopsis leaves in response to manipulation of the thiol-disulfide status. Feeding low concentrations of the sulfhydryl reagent dithiothreitol (DTT) for one hour at the end of the dark period led to post-translational redox-activation of ADP-glucose pyrophosphorylase and major alterations in leaf carbon partitioning, including an increased flux into major respiratory pathways, starch- cell-wall-, and amino-acid synthesis and a reduced flux to sucrose. This was accompanied by a decrease in the levels of hexose-phosphates, while metabolites in the second half of the TCA cycle and various amino acids increased, indicating a stimulation of anaplerotic fluxes reliant on α-ketoglutarate. There was also an increase in shikimate as a precursor of secondary plant products and marked changes in the levels of the minor sugars involved in ascorbate synthesis and cell wall metabolism. Transcript profiling revealed a relatively small number of changes in the levels of transcripts coding for components of redox-regulation, transport processes and cell wall, protein and amino acid metabolism, while there were no major alterations in transcript levels coding for enzymes involved in central metabolic pathways. These results provide a global picture of the effect of redox and reveal the utility of transcript and metabolite profiling as systemic strategies to uncover the occurrence of redox-modulation in vivo. Experiment Overall Design: Leaf discs were incubated with or without 5mM DTT for one hour to affect the redox status of the cells. The effect of this treatment on global gene expression was analysed using affymetrix ATH1 microarrays. The experiments contains one control (-DTT) and one treatment (+DTT) and is performed with 2 biological replicas.
Project description:Gene expression microarray was performed using early log phase culture at 37 degree and 200 rpm followed by RNA extraction from M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv For gene expression profiling, total RNA was isolated from wild type H37Rv, higB KO mutant strain and complemented strains. The bacterial pellets for M.tb H37Rv wild type, M.tb H37Rv HigB KO mutant strain and complemented strain were resuspended in 1.0 ml TRIzol (Invitrogen Corporation, Carlsbad, CA); mRNA was extracted as per standard protocols and cleaned using RNAeasy columns (Qiagen GmbH, Hilden, Germany). Extracted mRNA (1 μg) was treated with DNase I using the Ambion DNA-free kit (Invitrogen Corporation, Carlsbad, CA) to remove genomic DNA contamination and quantified using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The purity and integrity of RNA samples were assessed Agilent 2100 Bio analyzer (Agilent TechnologiesInc., USA). Further, 25ng of RNA was amplified and labeled using Low input Quick Amp WT Labeling kit (Agilent Technologies, USA). The labeled cRNA was purified using RNAeasy columns (Qiagen, USA) and total yields were quantified using Nanodrop 2000c spectrophotometer.
Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression.
Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression. Two color Experiment,Organism: Mycobacterium Tuberculosis, ilife Discoveries designed Custom Mycobacterium tuberculosis on 8x15k GE Microarray. Two-condition experiment, H37Rv vs. H37Rv/SL3. Biological replicates: 2 biological control H37RV replicates labelled with Cy3, 2 SL3 biological expressing replicates labelled with Cy5.