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

ABSTRACT: 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.

ORGANISM(S): Francisella tularensis

PROVIDER: GSE11555 | GEO | 2009/01/06

SECONDARY ACCESSION(S): PRJNA106285

REPOSITORIES: GEO

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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. all pairs

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