An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture. Sample "Dechlorinating_Community_DNA_ANAS_Rep2" was collected from the culture in March 2006.
Growth protocol
The Dehalococcoides-containing enrichment culture is grown and maintained in an anaerobic semi-batch reactor supplemented with modified-Tanner medium. Lactate (25 mM final concentration) is supplied as both the carbon source and electron donor, while TCE (0.1 mM final concentration) is supplied as the terminal electron acceptor.
Extracted molecule
genomic DNA
Extraction protocol
A 50-ml sample of the ANAS culture was collected after one 100- μM dose of TCE was degraded to VC and ethene. Cells were collected by centrifugation (12,000 x g for 3 minutes at 4°C), the supernatants were discarded, and the cell pellets were stored at –80°C until processing. ANAS genomic DNA (gDNA) was isolated from frozen cell pellets using an UltraClean Mega Prep Soil DNA kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. 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.
