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Generation of Ionic Liquid Tolerant Pseudomonas putida KT2440 Strains via Adaptive Laboratory Evolution

ABSTRACT: Transcriptome of the wildtype or evolved Pseudomonas putida KT2440

ORGANISM(S): Pseudomonas putida KT2440

PROVIDER: GSE149827 | GEO | 2020/07/08

REPOSITORIES: GEO

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Project description:In this study we exploited next-generation Illumina sequencing technology (Wang et al., 2009) to refine the current annotation of the KT2440 genome. Transcriptome sequencing data were queried for yet undescribed small RNAs and ORFs and employed to validate predicted operons and gene coordinates. Expression profiles were measured at 10Â°C and 30Â°C to cover the physiologic temperature profile of this mesophilic bacterium. Moreover we compared the sensitivity and specificity of transcriptome data by taking the same RNA preparations for cDNA sequencing and hybridization of Progenika and Affymetrix microarrays. This SuperSeries is composed of the SubSeries listed below. P. putida KT2440 (strain DSM6125) (Bagdasarian et al., 1981) was obtained from the German Resource Centre for Biological Material (DSMZ, (Braunschweig, Germany). Bacterial cultures were inoculated from a frozen stock culture of P. putida wild type, and incubated at 30Â°C for 8 hours at 250 rpm in LB medium. An aliquot of 0.2 mL was added to 20 mL M9 medium (Na2HPO4 33.9 g/L, KH2PO4 15.0 g/L, NaCl 2.5 g/L, NH4Cl 5.0 g/L, MgSO4 2 mM, CaCl2 0.1 mM, FeSO4 x 7 H2O 0.01 mM, pH 6.8) supplemented with 15 mM succinate as sole carbon source in a 100 mL flask and incubated overnight at 30Â°C. For RNA extraction, bacteria were grown in a 1.5 L batch culture (M9 + 15 mM succinate) using the BioFlo 110 Fermenter (New Brunswick Scientific Co., Edison, NJ) to ensure constant pH, aeration and agitation. When cultures reached mid-exponential phase (OD600 ~ 0.8) the temperature was decreased from 30Â°C to 10Â°C. Three samples, each for parallel RNA extraction, were subsequently taken immediately before temperature downshift (30Â°C) and two hours after the media had been cooled to 10Â°C. The gene expression profiles of P. putida KT2440 at 30Â°C and 10Â°C were analyzed using three different transcriptome platforms: RNA-Seq (Illumina cDNA sequencing), Affymetrix microarrays and Progenika oligonucleotide microarrays. For a detailed description of sample preparation (RNA, cDNA , labelling, hybridization etc.) refer to individual Series.

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Generation of Ionic Liquid Tolerant Pseudomonas putida KT2440 Strains via Adaptive Laboratory Evolution

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