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Vibrio cholerae Freezing Study

ABSTRACT: Question Addressed: Does gene expression in V. cholerae change when samples are frozen at -80 degrees? An in vitro grown culture of O1 Inaba ICDDR,B (strain DSM-V999 described in Nature. 2002 Jun 6;417(6889):642-5) that had been grown to exponential phase (OD600 + 0.2) was placed in a 15 ml conical and then placed in a -80 degree freezer. A portion of the starting culture was harvested prior to freezing to serve as the control/reference for downstream hybridizations. RNA was recovered from the non-frozen sample as well as by prepping material directly from frozen samples. Labeling reactions were performed in quadruplicate for each sample. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments.

ORGANISM(S): Vibrio cholerae

PROVIDER: GSE38179 | GEO | 2013/05/23

SECONDARY ACCESSION(S): PRJNA167440

REPOSITORIES: GEO

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Vibrio cholerae Freezing Study

Project description:Question Addressed: Does gene expression in V. cholerae change when samples are frozen at -80 degrees? An in vitro grown culture of O1 Inaba ICDDR,B (strain DSM-V999 described in Nature. 2002 Jun 6;417(6889):642-5) that had been grown to exponential phase (OD600 + 0.2) was placed in a 15 ml conical and then placed in a -80 degree freezer. A portion of the starting culture was harvested prior to freezing to serve as the control/reference for downstream hybridizations. RNA was recovered from the non-frozen sample as well as by prepping material directly from frozen samples. Labeling reactions were performed in quadruplicate for each sample. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. replicate_design

2013-05-23 | E-GEOD-38179 | biostudies-arrayexpress

Mixed biofilm - cluster transfer O1 to O139

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Project description:These experiments were performed to show a serogroup conversion in Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 WT = A1552 was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (2 ug each) from strain VC73-orf6/7-Kan-A was added after 24h and the cells grown further for 24h. The VC73-orf6/7-Kan-A strain is a ATCC25873 derivative (both O37 serogroup) which harbors a Kanamycin cassette in the O37 region (as part of the operon between orf6 and orf7 w/o own promotor) for better selection. Transformants were selected on LB+Kan plates. Three clones were selected from each experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Comparison of A1552 versus VC73-orf6/7-Kan-A is shown as control. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Keywords: genotyping_design

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Project description:These experiment were performed to show a serogroup conversion in Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 WT = A1552 was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (2 ug each) from strain VCO139-Kan was added after 24h and the cells grwon further for 24h. The VCO139-Kan strain is a MO10 derivative (both O139 serogroup) which harbors a Kanamycin cassette in the O139 region (as part of the operon between wbfA and wbfB w/o own promotor) for better selection. Transformants were selected on LB+Kan plates. Two groups of transformants were gained: Group I had a full exchange of the O1 region by the O139 region (clones serogroup-converted: SGC#1-3); the crossovers for the homologous recombination event had occurred within or upstream of the gmhD gene and in most instances within or downstream of the homolog gene of VC0271. This implies an exchange of an at least 33 kb spanning O1 genomic region by more than 42 kb of the O139 region. Group II had only half of the O139 region transfered and therefore half of the O1 region kept (clones HSGC#4-6). We analyzed their genotype and found that all of them had undergone a homologous recombination event with one crossover in or upstream of the gmhD gene and the second one inside the VC0254 and IS1358 gene. The transformation experiment was done three independent times (I - III). Three clones from group I and group II were selected from each experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Strain Name: -III for experimental replicates (transformation); clones #1 -#3 full region exchanged; clones #4-#6 half region exchanged. Two MA replicates for each clone Keywords: genotyping_design, array CGH

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