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Phosphomannose isomerase gene (PMI40) deletion strain cultivated in varying initial mannose concentrations

ABSTRACT: YEAST STRAIN: A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9). CULTIVATION MEDIUM: The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 × 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 × 7 H2O, 0.84 g/l MnCl2 × H2O, 0.3 g/l CuSO4 × 5 H2O, 3 g/l FeSO4 × 7 H2O (Riedel-de-Haën AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 × 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 × 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 × 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA). The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L). CULTIVATIONS AND SAMPLING: Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 °C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0. In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation. MEASUREMENT OF GENE EXPRESSION LEVELS: 0.9 mg CDW of cells were quenched in cold (-40 °C) methanol (45%) and centrifuged (1 min, +4 °C, 16100 G). The pellets were suspended in 400 µl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 µl 1:1 phenol-chloroform, and 5 µl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 °C. The mixtures were centrifuged (15 min, +4 °C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 µg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates. Keywords: dose response

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE1938 | GEO | 2004/11/10

SECONDARY ACCESSION(S): PRJNA90429

REPOSITORIES: GEO

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Project description:YEAST STRAIN:,A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9).,CULTIVATION MEDIUM:,The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 Ã 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 Ã 7 H2O, 0.84 g/l MnCl2 Ã H2O, 0.3 g/l CuSO4 Ã 5 H2O, 3 g/l FeSO4 Ã 7 H2O (Riedel-de-HaÃ«n AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 Ã 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 Ã 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 Ã 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA).,The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L).,CULTIVATIONS AND SAMPLING:,Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 Â°C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0.,In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation.,MEASUREMENT OF GENE EXPRESSION LEVELS:, ,0.9 mg CDW of cells were quenched in cold (-40 Â°C) methanol (45%) and centrifuged (1 min, +4 Â°C, 16100 G). The pellets were suspended in 400 Âµl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 Âµl 1:1 phenol-chloroform, and 5 Âµl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 Â°C. The mixtures were centrifuged (15 min, +4 Â°C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 Âµg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates.

Project description:The comprehensive transcriptome of Streptomyces lividans TK24 in a biolector milliliter-scale cultivation system was compared in detail against an established 1000-fold larger lab-scale bioreactor system. For main-cultures, the modified minimal medium NMMP (D'Huys et al. 2011) was applied, containing 2.07 g/L NaH2PO4 x 1 H2O, 2.6 g/L K2HPO4, 0.6 MgSO4 x 7 H2O, 3.0 g/L (NH4)2SO4, 10 g/L D-glucose and 5 g/L BactoTM Casamino acids as well as several trace elements (1 mg/L ZnSO4 x 7 H2O, 1 mg/L FeSO4 x 7 H2O, 1 mg/L MnCl2 x 4 H2O, 1 mg/L CaCl2). pH was adjusted to 6.8 using 1 M KOH or 1 M H2SO4. In all MTP BioLector (BL) cultivations and in some bioreactor experiments (when indicated) 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) was added as biological inert buffer system. Except for the phosphate buffer, all other components were prepared as stock solutions and sterilized separately. For MTP based cultivations the BL device, all stock solutions kept frozen at -20 C in aliquots sufficient for 50 mL final medium, to ensure identical conditions between experimental runs. For the system comparison experiments the same medium charge was used for all cultivations. 1 mL MTP-based BL cultivations MTP cultivations were performed in 48 well FlowerPlates (m2p-labs GmbH, Baesweiler, Germany), covered by a gas-permeable sealing foil (m2p-labs GmbH, Baesweiler, Germany). 1000 L cultivation medium was inoculated to a final OD600 of 0.2. Temperature and humidity was controlled in the incubation chamber of the BL device at 30 C and 89 % respectively. All signals (BS, DO, and pH), were measured with a cycle time of around 10 min. Altogether, 96 identical parallel milliliter-scale cultivations were performed in two separate but identical BL devices. One of these devices was additionally embedded in a lab-robotic liquid handling unit in the style of (Unthan et al. 2015; Rohe et al. 2012), to provide automated hourly sampling for substrate and cell dry weight analysis. The cultivations in the second BL unit served to generate samples for the omics-based analysis, discussed in the later sections. Bioreactor cultivations were carried out in a 1.5 L lab-scale system (DasGip, Jlich, Germany), equipped with two six blade, 46 mm Rushton turbines as well as sensors for DO (Hamilton, Visiferm DO 225) and pH (MettlerToledo, 405-DPAS-SC-K8S/225/120). Temperature was controlled at 30 C; evaporation was minimized by offgas cooling at 10 C. 700 mL phosphate buffer was sterilized directly within the reactor glass vessel; all other media components were added sterile and premixed subsequently by using a 500 mL feeding flask. 0.5 mL anti-foam (AF204 (Sigma, MO, USA)) was added before DO calibration. The final 1000 mL medium was inoculated with washed mycelium to a final OD600 of 0.2. Both, aeration and agitation were set to fixed values at 0.5 vvm and 800 rpm respectively. For both techniques samples were taken during the late growth phase, two hours after the switch to process phase II.

Project description:Purpose: We aim to identify transciptional changes of human CD4+/CD8+ T cells due to high fat low carbohydrate ketogenic diet (KD) in vivo. Method: Healthy volunteers conducted a 21 days ketogenic diet, limiting carbohydrate intake to <30g/day. Before the start (T0) and at the end (T1) of the diet, blood samples were taken and PBMC were isolated. PBMCs were obtained by density centrifugation (Histopaque 1077, Sigma-Aldrich, St. Louis, MO, USA). A ViCell analyzer (Beckman Coulter, Fullerton, CA, USA) was used to evaluate the cell count and viability. Only samples exceeding a cell viability of 90% were processed further. PBMCs were subjected to cell cultivation in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany), 1% HEPES (Sigma-Aldrich, St. Louis, MO) and 1% L-glutamine (Life Technologies, Carlsbad, CA, USA). T cells were stimulated via the addition of CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) with a bead-to-cell ratio of 1:8 for a duration 24 hours. After stimulation, CD3/CD28 Dynabeads were magnetically removed. Pan T cell-, CD4+- and CD8+-cell-isolation was performed by magnetic cell separation (Pan T Cell Isolation Kit, # 130-096-535 | human CD4 MicroBeads, # 130-045-101 | human CD8 MicroBeads, # 130-045-201, Miltenyi Biotec, Bergisch Gladbach, Germany) using an AutoMACS Pro Separator ( # 130-092-545, Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. Results: 11.545 expressed genes were identified, for CD4/CD8 T cells, we detected 5.667/ 5.799 up-regulated genes and 5.878/5.746 down-regulated genes. 294/346 genes and 325/252 genes were significantly up/down-regulated for CD4+/CD8+ T cells (p-val. <0.05). Gene set enrichment analysis revealed 117/17 and 22/6 significantly up/down-regulated pathways for CD4+/CD8+ T cells (p-val. <0.05). Genes and Gene sets differentially regulated were relevant for T cell immune response and metabolic function. Conclusion: KD resulted in immunometabolic reprogramming of human CD4+/CD8+ T cells.

Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

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Phosphomannose isomerase gene (PMI40) deletion strain cultivated in varying initial mannose concentrations

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