Pure culture Dehalobacter restrictus collected in March 2006 was used as a negative control for the experiment.
Growth protocol
Dehalobacter restrictus PER-K23 (DSMZ strain number 9455) was grown in 100-ml batch pure cultures with a defined mineral salts medium containing 5mM acetate as the carbon source, H2 as the electron donor (added as H2:CO2 gas [80:20 vol:vol]), and 78 μmol TCE as the electron acceptor.
Extracted molecule
genomic DNA
Extraction protocol
Dehalobacter restrictus cells were harvested from ten 100-ml batches per culture to assure that a sufficient quantity of DNA could be extracted for application to microarrays. Cells were collected by vacuum filtration with hydrophilic Durapore membrane filters (0.22 µm pore size, 47 mm diameter [Millipores, Billerica, MA]), and the filters were stored in 2-ml microcentrifuge tubes at –80°C until further processing. gDNA was isolated from filters using a phenol extraction method as follows. The 2-ml microcentrifuge tubes containing frozen filters were amended with 250 µl lysis buffer (200 mM Tris [pH 8.0], 50 mMEDTA, and 200 mM NaCl), 100 µl 10% sodium dodecyl sulfate, 1 g 100-µm-diameter zirconia-silica beads (Biospec Products, Bartlesville, OK), and 1.0 ml buffer-equilibrated phenol (pH 8.0) (Sigma-Aldrich, St. Louis, MO). Cells were lysed by heating the tubes to 65°C for 2 min, bead beating them with a Mini Bead Beater (Biospec Products) for 2 min, incubating for 8 min at 65°C, and bead beating again for an additional 2 min. Cellular debris was collected by centrifugation (12,000 x g for 5 minutes at 4°C), and the aqueous lysate was transferred to a new DNase-free microcentrifuge tube. The aqueous lysate was extracted twice with 1 volume of phenol (pH 8.0)-chloroform-isoamylalcohol (125:24:1 vol:vol) and once with 1 volume of chloroform-isoamylalcohol (24:1 vol:vol) (Sigma-Aldrich). DNA was precipitated by adding 0.1 volume of 3 M ammonium acetate (pH 5.2) and 1 volume of 100% isopropanol. The precipitate was collected by centrifugation, washed once with 80% ethanol, and resuspended in 40 µl of nuclease-free water. Contaminating RNA was removed by RNase digestion with DNase-free RNase according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). The purified DNA was stored at –80°C prior to further use.
Label
biotin
Label protocol
gDNA was prepared for application to microarrays with minimal modifications to the protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Briefly, purified gDNA (1 μg per array) was mixed with the positive control plasmids (7.6 nM lys, 15.2 nM phe, 30.4 nM thr, and 114.0 nM dap) described above and fragmented to an average size of 50 to 200 bp by enzymatic digestion with amplification grade DNase I (Invitrogen Life Technologies, Carlsbad, CA). Each 50-μl fragmentation reaction consisted of gDNA and 0.02 units DNase I per μg DNA diluted in 1 x One-Phor-All Buffer (Amersham Biosciences). The mixture was incubated for 20 minutes at 25°C, followed by 10 minutes at 98°C to inactivate the DNase I. The fragmentation products were visualized on a 4% MetaPhor Agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME) electrophoresis gel run at 70 V for 2 hours. 25 µl of the fragmentation product was then used for further processing. The fragmentation products were biotin end-labeled by adding 25 µl of fragmentation product to 10 µl of 5 x reaction buffer (Affymetrix, Santa Clara, CA), 2 µl 7.5 mM GeneChip DNA labeling reagent (Affymetrix), 2 µl terminal deoxynucleotidyl transferase (Promega), and 11 µl nuclease-free water, incubating at 37°C for 60 minutes, and adding 2 µl 0.5 M EDTA (pH 8 .0) (Invitrogen Life Technologies, Carlsbad, CA) to stop the reaction.
Hybridization protocol
gDNA was prepared for application to microarrays with minimal modifications to the protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Microarray chips were prehybridized with 1 x hybridization buffer [100 mM MES (Sigma-Aldrich), 1 M [Na+] (Sigma-Aldrich), 20 mM EDTA (Sigma-Aldrich), 0.01% Surfact-Amps Tween-20 (Pierce Chemical)] in a chip-rotisserie oven for at least 20 minutes at 60 rmp and 45°C. The samples were denatured by heating to 99°C for 5 minutes. Hybridization buffer was removed from each microarray chip and replaced with an equal volume of sample hybridization mixture (1 x MES hybridization buffer, 7.8 % DMSO [Sigma-Aldrich], 0.1 mg/ml herring sperm DNA [Promega Corporation], 0.5 mg/ml BSA [Invitrogen Life Technologies, Carlsbad, CA], 50 pM B2 control oligonucleotide [Affymetrix], and 1 µg fragmented labeled DNA). Hybridization was carried out by incubating in a chip-rotisserie oven (Affymetrix) for 16 hours at 45°C and 60 rmp. Chips were washed and stained according to the Affymetrix protocol using the Affymetrix GeneChip fluidics station.
Scan protocol
Affymetrix chips were scanned according to the protocols outlined in section 3, chapter 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA).
Description
Positive control probe sets target Bacillus subtilis genes lys, phe, thr, and dap. To generate control DNA, plasmids containing lys, phe, thr, and dap were purified from strains ATCC87482, ATCC87483, ATCC87484, and ATCC87486, respectively. These plasmid standards were spiked into each sample before fragmentation. Negative control probe sets were designed to hybridize with specific Escherichia coli and bacteriophage genes that were not expected to be present in experimental cultures.
Data processing
The hybridization signal intensities for each probe set was computed using Affymetrix GeneChip Operating Software and the MAS5 algorithm. The data set of each microarray was normalized by scaling the signal intensities of the added positive controls to a target signal intensity of 2500 and adjusting all other probe set signal intensities accordingly with respect to the scaled positive control signal intensities, thus allowing for comparisons between microarray chips.
Comparative Genomics of D. ethenogenes 195 and an Unsequenced Dehalococcoides-Containing Enrichment Culture
Data table header descriptions
ID_REF
VALUE
The hybridization signal intensities for each probe set was computed using Affymetrix GeneChip Operating Software and the MAS5 algorithm. The data set of each microarray was normalized by scaling the signal intensities of the added positive controls to a target signal intensity of 2500 and adjusting all other probe set signal intensities accordingly with respect to the scaled positive control signal intensities, thus allowing for comparisons between microarray chips.
