Project description:Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM. In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium. Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagenâ??s RNeasy Mini kit (using the supplierâ??s protocol) prior to use in microarray analysis. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Cells were grown in glycerol-glycerophosphate medium (GGM). Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM),beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), ZnO (6.14 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with or without Zn as necessary) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above. All chemicals were of AnalaR grade of purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm Ã? 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d. E. coli strain MG1655 was grown in parallel custom-built chemostats made entirely of non-metal parts. One chemostat was fed medium that contained â??adequateâ?? Zn (that normally found in GGM), whilst the other contained no added Zn and had been actively depleted of Zn by use of the special precautions listed here. Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water. Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing. Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen). Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel. The dilution rate (and hence the specific growth rate) was 0.1 h-1. The vessel was inoculated using one of the side-arms. Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400). Stirring speed was calibrated (437 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd). Temperature was kept constant at 37Â°C. Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates. The â??Zn-freeâ?? chemostat was inoculated with cells that had been sub-cultured in Zn-free medium to deplete internal stores of Zn. A 0.25 ml aliquot of a saturated culture of strain MG1655 grown in LB was centrifuged and the pellet used to inoculate 5 ml of GGM that was incubated overnight at 37Â°C with shaking. A 2.4 ml (i.e. 2% of chemostat volume) aliquot of this was then used to inoculate the chemostat. The â??adequate Znâ?? chemostat was inoculated with cells treated in essentially the same way but grown in GGM containing an â??adequateâ?? level of Zn. The two cultures (+/- Zn) used to inoculate the chemostats had OD600 readings within 2.5% of each other. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, 10 ml samples were removed from the chemostats and total RNA was stabilised using RNAprotect (Qiagen). Total RNA was purified using Qiagenâ??s RNeasy mini kit. Equal quantities of RNA from adequate Zn and Zn-limited chemostats were labeld by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labeld with Cy3-dCTP, while the other sample was labeld with Cy5-dCTP. RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65Â°C for 10 min, followed by 10 min at 22Â°C. This was supplemented with 4 microlitres of 5Ã? First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42Â°C for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labeld sample was mixed with the Cy5-dCTP-labeld sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95Â°C for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 Â°C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 Â°C) SSC buffers for 5 min each at 37Â°C: 1Ã? SSC/0.1 % SDS, 1Ã? SSC, 0.2Ã? SSC and 0.01Ã? SSC. Microarray slides were dried by centrifugation at 500 Ã? g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 or Cy5/Cy3 (test/reference) fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of < 0.05 (as determined by a t-test) were defined as being statistically significantly differentially transcribed.

Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 ÂµM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 ÂµM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 ÂµM EDTA, 31 ÂµM FeCl3, 6.15 ÂµM ZnO, CuCl2 0.57 ÂµM, CoNo3 0.34 ÂµM, 1.6 ÂµM H3BO3, 1 ÂµM ammonium molybdenate and 1 ÂµM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 ÂµM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5Ã First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 Â°C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 Â°C) SSC buffers for 5 min each at 37oC: 2Ã SSC/0.1 % SDS, 1Ã SSC, 0.2Ã SSC and 0.01Ã SSC. Microarray slides were dried by centrifugation at 1500 Ã g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â¥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Project description:The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays. S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study. Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0. The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity. Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus. Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis). Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell. Keywords: Immunlogical response to IHN virus challenge A preliminary microarray experiment was run on the waterborne-challenged S. salar whereby the five individuals sampled on a given day were combined and run on a single slide, and we compared the expression profiles from each day (days 0 (unchallenged control) through 11). From this experiment, we noted that the most profound changes were occurring around days 1, 5, and 9, so we performed subsequent experiments at these time points, with five fish per day and 5 control fish analysed in total.

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