Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq system comparison of Streptomyces lividans TK24 cultivated under micro-scale biolector and reference bioreactor conditions

ABSTRACT: 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.

INSTRUMENT(S): Illumina HiSeq 1500

ORGANISM(S): Streptomyces lividans

SUBMITTER: Tobias Busche

PROVIDER: E-MTAB-5047 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 × 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

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:We used a comprehensive quantitative proteomic profiling based on LC-MS/MS combined with TMT labeling strategy to analyze frozen-thawed Ashidan yak spermatozoa proteomic dynamic changes under three sequential conditions: density gradient centrifugation-based purification（FTH sperm）, incubation in capacitation medium(CAP sperm), and treatment with the calcium ionophore A23187 to induce the acrosome reaction process(AR sperm). FTH sperm Briefly, sperm-containing straws were thawed for 30 s at 37ºC prior to addition to a prepared 45-90% Percoll solution (Percoll P1644; 3.1mM KCl, 2.9mM NaH2PO4,80mM NaCl, 10mM HEPES, 2mM CaCl2·H2O, 0.4mM MgCl2·6H2O, 26mM Sodium DL-Lactate Solution, 25mM NaHCO3; pH 7.3-7.4) and diluted using modified Tyrode's Hepes buffered medium (Sp-TALPH, 114mM NaCl, 3.2mM KCl, 2mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 10mM HEPES, 3mg/ml BSA, 0.2mM Na pyruvate, 7.5x10-3mg/ml Gentamicin; pH 7.3-7.4) followed by centrifugation for 10 min at 700 xg to enable the isolation of highly motile sperm. To ensure that the gradient material was completely eliminated from these samples, the sperm cells collected from the bottom layer were washed two times using supplemented Sp-TALPH medium and centrifuged for 5 min at 300 xg. Sperm were then isolated and adjusted to a concentration of 100x106/mL using modified Tyrode's bicarbonate buffered medium (Sp-TALP; 114mM NaCl, 3.2mM KCl, 25mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 6mg/ml BSA, 0.2mM Na pyruvate, 5x10-3mg/ml Gentamicin; pH 7.3-7.4). A CASA approach was then used to assess sperm motility, with only those samples exhibiting >70% total motility being retained for further analyses, as per previously published protocols. An aliquot containing 100 million FTH sperm was taken from each sample for subsequent protein isolation, with the remaining sperm being incubated in capacitation medium. CAP sperm Capacitation medium was composed of Sp-TALP containing heparin (50 ug/ml) and caffeine (5 mM), and sperm were incubated in this medium for 30 min at 38.5ºC in a 5%CO2 incubator, as these conditions have previously been shown to be optimal for the induction of capacitation in frozen-thawed yak sperm. An aliquot containing 100 million CAP sperm was taken from each sample for subsequent protein isolation. AR sperm AR induction was achieved by treating 990 uL capacitated sperm samples with 10 µL of A23187 (1.0mM in DMSO, CSNpharm, IL, USA) to a final concentration of 10 µM, followed by a 15 min incubation at 38.5ºC, For individual replicates, aliquots of 100 million AR sperm were collected for subsequent protein analyses.

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RNA-seq system comparison of Streptomyces lividans TK24 cultivated under micro-scale biolector and reference bioreactor conditions

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