Serine hydrxamate (100 mg/L final concentration) was added to the cultures in the late-exponential phase at ~11 OD (~5.5 g g dry cell weight per liter). Samples for DNA microarray analysis were taken 1 hour after the beginning the serine hydrxamate addition (13.2 OD, ~6.6 g dry cell weight per liter, growth rate 0.15 1/h).
Growth protocol
The strain Escherichia coli MG1655 [pPROEx-CAT] was cultured in a fed-batch fermenter in LB media w/ glucose and a glucose/MgS04 feed at 37 degrees. Cultures were synchronized to the late log phase, approximately 4.5 hours of fermentation time. Serine hydrxamate (100mg/L final concentration) was added to the cultures. Samples were taken 1 hour after the beginning the serine hydrxamate addition.
Extracted molecule
total RNA
Extraction protocol
Fifteen mL fermentation broth was used for each RNA extraction. Harvested cells were rapidly cooled in a -80°C ethanol bath, then centrifuged at 5000 g for seven minutes at 4°C. The supernatant was discarded. The cell pellet was frozen at -80°C. Thawed (on ice) cell pellets were suspended in 150 mL of TE buffer (10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA) containing 0.75 mg/mL lysozyme, and incubated for 15 minutes at room temperature. Total RNA was isolated using RNAqueousTM-Midi Kit (Ambion). The concentration of purified total RNA was determined spectrophotometrically by measuring absorbance at 260 nm. The quality and integrity of the isolated RNA was confirmed by agarose gels (clear 23S and 16S rRNA bands).
Label
biotin
Label protocol
The cDNA synthesis was conducted as described in Affymetrix Expression Handbook (Affymetrix Inc., Santa Clara, CA). Briefly, 10 μg of purified total RNA was mixed with 750 ng of random primers (Invitrogen Life Technologies) and incubated at 70°C for 10 minutes. The samples were cooled to 25°C and incubated for 10 minutes. RNA/Primer hybridization mixture was mixed with dNTPs (Amersham Pharmacia Biotech), Superscript II reverse transcriptase and 5X 1st Strand Buffer (Invitrogen Life Technologies), SUPERase•ln (Ambion) and DTT. The cDNA synthesis mixture was incubated at 37°C for 60 minutes, then incubated at 42°C for 60 minutes. The SuperScript II was heat inactivated by incubation at 70°C for 10 minutes. The mixture was then cooled to 4°C. RNA was degraded by the addition of 20 μL 1N NaOH. The samples were incubated at 65°C for 30 minutes and then neutralized by 20 μL 1N HCl. The cDNA was purified using QIAquick Columns (QIAquick® Spin Kit, QIAGEN). cDNA product was eluted with 60 μL of elution buffer supplied with the QIAquick Kit. Purified cDNA product was quantified by absorbance at 260 nm. Typical yields were 4-7 μg cDNA. Four micrograms of purified cDNA was mixed with DNase I and 10X One-phore-All Buffer (Amersham Pharmacia Biotech). The mixture was incubated at 37°C for 10 minutes followed by inactivation of the DNase I at 98°C for 10 minutes. Fragmentation was confirmed by separation of the cDNA on 4-20% acrylamide gels (BioRad) stained with SYBR Gold (Molecular Probes). The majority of the DNA fragments after fragmentation were between 50-200 bases. The fragmented cDNA was 3′-end-labeled using the Enzo® BioArrayTM Terminal Labeling Kit (Affymetrix Inc., Santa Clara, CA). Briefly, 5X reaction buffer, 10X CoCl2, 100X Biotin-ddUTP and 50X terminal deoxynucleotides transferase were mixed and incubated at 37°C for 60 minutes. The reaction was stopped by the addition of 2 μL 0.5 EDTA. The product was stored at -20°C until the hybridization step.
Hybridization protocol
Hybridization of the fragmented cDNA with the Affymetrix Antisense E. coli GeneChip microarrays were conducted according to the manufacturer's instructions. Briefly, samples were hybridized with the array in a solution containing 100 mM MES buffer, 1 M NaCl, 20 mM EDTA and 0.01% (v/v) Tween 20, pH 6.6 (1X MES). Additionally, the solution contained 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA and 3.09 μg fragmented-labeled cDNA. The hybridization cocktail (200 μL) was added to the probe array at room temperature. The probe array was placed in a rotary hybridization oven set at 60 rpm at 45°C for 16 hours. Following hybridization, the sample solution was removed and saved. The probe array was washed and stained automatically by the fluidization station (Affymetrix Inc.) as recommended in the technical manual (Affymetrix, Inc.). The signals were enhanced with a Streptavidin solution (1X MES stain buffer, 2 mg/mL BSA, 10 μ/ mL streptavidin ). After the streptavidin solution was removed, an antibody solution (1X MES stain buffer, 2 mg/mL, 0.1 mg/mL normal goat IgG and 5 μg/mL Biotin Anti-streptavidin) was added. Streptavidin phycoerythrin solution (1X MES stain buffer, 2 mg/mL, 10 μg/mL streptavidin-phycoerythrin) was used to fluorescently label the arrays.
Scan protocol
The wash and stain procedures were carried out by the fluidics station using the ProkGE-fluidics script (Affymetrix Inc.).
Description
38 FK2-2
Data processing
Microarray suit 5.0 (Affymetrix Inc.) was used to average the scans and process the data, further data analysis was conducted using Affymetrix® Data Mining Tool (DMT 3.0).
Serine hydroxamate and the transcriptome of high cell density recombinant E. coli MG1655
Data table header descriptions
ID_REF
VALUE
The raw intensities (per gene) were normalized to the median array intensity and set to a constant value (150). GeneSpring software was used for ANOVA analysis.
