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Transcriptional profiling of wild-type, mglA point mutant, and fevR (FTT0383) knockout bacteria

ABSTRACT: Time courses and RNA isolation. Overnight cultures of F. novicida strain U112, GB2, and fevR ko (383) were grown in TSB 0.2% cysteine at 37C with shaking and subcultured to an OD600 of 0.01 in 200ml. Samples were taken throughout the growth curve for RNA isolation and to determine the OD600. Five samples were taken during the growth curve at 1, 3, 5, and 7.5h and 10h for characterization of single mutants or at 2, 4, 6, and 8h in gro::fevR bypass experiments. RNA was harvested and a common reference was created from each individual sample. 1ug of either the Sample, or Reference was reverse transcribed and labeled with either Cy5 (samples) or Cy3 (reference) and hybridized to a Francisella microarray previously described in Brotcke, Weiss, et al, Infection and Immunity, 2006. Groups of assays that are related as part of a time series. Age: Red channel represents 1ug of RNA from an individual timepoint Starting OD: Wt, GB2, and FTT0383 (fevR) ko were subbed to an OD of 0.01 in TSB + 0.2% cysteine in 200ml. Keywords: time_series_design

ORGANISM(S): Francisella tularensis

PROVIDER: GSE11554 | GEO | 2009/01/06

SECONDARY ACCESSION(S): PRJNA106283

REPOSITORIES: GEO

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Transcriptional profiling of wild-type, mglA point mutant, and fevR (FTT0383) knockout bacteria

Project description:Time courses and RNA isolation. Overnight cultures of F. novicida strain U112, GB2, and fevR ko (383) were grown in TSB 0.2% cysteine at 37C with shaking and subcultured to an OD600 of 0.01 in 200ml. Samples were taken throughout the growth curve for RNA isolation and to determine the OD600. Five samples were taken during the growth curve at 1, 3, 5, and 7.5h and 10h for characterization of single mutants or at 2, 4, 6, and 8h in gro::fevR bypass experiments. RNA was harvested and a common reference was created from each individual sample. 1ug of either the Sample, or Reference was reverse transcribed and labeled with either Cy5 (samples) or Cy3 (reference) and hybridized to a Francisella microarray previously described in Brotcke, Weiss, et al, Infection and Immunity, 2006. Groups of assays that are related as part of a time series. Age: Red channel represents 1ug of RNA from an individual timepoint Starting OD: Wt, GB2, and FTT0383 (fevR) ko were subbed to an OD of 0.01 in TSB + 0.2% cysteine in 200ml. Keywords: time_series_design Computed

2009-01-05 | E-GEOD-11554 | biostudies-arrayexpress

Constitutive Expression of FevR (383) in wt, mglA mutant, and FevR mutant bacteria

Project description:Time courses and RNA isolation. Strains were created that constitutively express fevR (FTT0383) transcript by replacing the endogenous fevR promoter with that of groES, and this gro::fevR region was integrated into either the wild-type, mglA mutant (GB2), or fevR knockout. Overnight cultures of F. novicida strains were grown in TSB 0.2% cysteine at 37C with shaking and subcultured to an OD600 of 0.01 in 200ml. Samples were taken throughout the growth curve for RNA isolation and to determine the OD600. Four samples were taken during the growth curve at 2, 4, 6, and 8h for characterization of in gro::fevR bypass experiments. RNA was harvested and a common reference was created by combining equal amounts of RNA from each sample in the experiment. 1ug of RNA from either the samples or common reference was reverse transcribed and samples were labeled with Cy5 (samples) or Cy3 (reference) and hybridized to the Francisella microarray described in Brotcke, Weiss, et al. Infection and Immunity, 2006. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

2009-01-06 | GSE11555 | GEO

Constitutive Expression of FevR (383) in wt, mglA mutant, and FevR mutant bacteria

2009-01-05 | E-GEOD-11555 | biostudies-arrayexpress

Global mapping of RNA-binding proteins in multi-drug resistant Staphylococcus aureus using 2C_2

Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.

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Halobacterium NRC-1 Growth curve and GTF perturbation

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Characterization of mycobacterial oligopeptide permease system

Project description:M. tuberculosis strain 1254 wild type (WT) and its isogenic delta Rv3662c-Rv3665c KO (opp KO) and complemented opp::opp were cultured in vitro, in liquid 7H9 media supplemented with OADC and 0.05% Tween 80, 0.5% glycerol, in a 150 mL volume, contained in roller bottles, incubated at 37oC. Growth was monitored as OD600 nm and recorded on a daily basis. In all experiments, the starting OD600 nm was 0.04-0.05. Aliquots were removed at each OD600 nm to make serial dilutions, plate onto 7H10 OADC agar plates for CFU counts. When cells reached OD600 nm of 0.3, 1.0, and 1.5, a 10-30 mL aliquot was taken, centrifuged 3 min at 3000 g, at room temperature, and the supernatant was discarded, with cell pellets being immediately frozen by inclusion into dry ice. Frozen cell pellets were stored at -80oC for later RNA isolation. In all experiments, RNA from WT was labeled with Cy5, and RNA from the opp KO was labeled with Cy3. In all experiments, the microarray comparsion used RNA from cells of the same growth stage, i.e., WT OD 0.3 vs opp KO OD 0.3, WT OD 1.0 vs opp KO OD 1.0, and WT OD 1.5 vs opp KO OD 1.5. When comparing the Complemented opp::opp vs the mutant opp KO, RNA from the later strain was Cy3-labeled. A cell type comparison design experiment design type compares cells of different type for example different cell lines.

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Pyrococcus furiosus DSM 3638 growth curve, tiling arrays

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