strain description: Mixed microbial community containing Dehalococcoides, Geobacter,
Growth protocol
Experiments were set up to examine gene expression in KB1 during vinyl chloride degradation using a robust and stable KB1 enrichment that had been maintained on TCE and MeOH for 5 years (T3MP1). This culture routinely degrades 200 µmoles of TCE to ethene every week. For each experiment the T3MP1 culture was purged with N2/CO2 (80:20 vol%) to remove trace chlorinated compounds and left to starve for four days to remove mRNA. Subsequently, 1.2 L of culture was split into two equal treatments of 600 mL in 1L bottles (Pyrex, VWR, Mississauga, ON) in an anaerobic glovebox (Coy Laboratory products, Grass Lake, MI). Into one treatment 5 mL (200 µmoles) of vinyl chloride was added as the electron acceptor and 15 µL (370 µmoles) methanol as the electron donor (VC+MeOH treatment). In the other treatment only the electron donor methanol was added (MeOH only). For the first set of arrays, RNA was extracted from the MeOH only and VC+MeOH treatments after 3 hours when 35 µmoles of ethene had been produced in the VC+MeOH treatment. A second set of experiments was performed a month later with the same set-up. For these, RNA was extracted after 3.5 hours when 50 µmoles of ethene had been produced in the VC+MeOH treatment. Within each biological set there were dye-switched duplicates, and technical replicates resulting in 6 arrays comparing the same treatments.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, 50 ml samples were withdrawn from the culture bottles inside an anaerobic chamber. After lysis with SDS and bead beating RNA was extracted using an ice-cold acid-phenol:chloroform:isoamyl alcohol method(Waller et al., 2005). The RNA solution was further purified using an RNeasy spin column (Qiagen,Valencia, California), and contaminating DNA was removed using the DNA-free kit (Ambion, Austin, TX). The quantity of the RNA was assessed using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the quality was assessed by electrophoresis on a 1% agarose gel or using the 2100 Bioanalyzer (Agilent Technologies).
Label
Cy3
Label protocol
The RNA was labelled using an indirect method in which amino allyl-dUTPs were incorporated during reverse transcription (RT), and then monoreactive Cyanine dyes were coupled to the aminoallyl cDNA in a separate reaction. First strand cDNA synthesis was carried out using 10-20 μg of total RNA, and 10 ng control RNA (Arabadopsis chlorophyll synthetase gene). The Cy3 or Cy5 labelled cDNA was purified then quantified using the NanoDrop ND-1000 to ensure that an appropriate amount of dye was incorporated (>100 pmoles) at an appropriate frequency (25-50 nucleotides per dye molecules). The labeled-cDNA was then hybridized to the slide at 37 0 C for 16-18 hours. The slides were then washed, dried and scanned with an Axon scanner using GenePix Pro software.
strain description: Mixed microbial community containing Dehalococcoides, Geobacter,
Growth protocol
Experiments were set up to examine gene expression in KB1 during vinyl chloride degradation using a robust and stable KB1 enrichment that had been maintained on TCE and MeOH for 5 years (T3MP1). This culture routinely degrades 200 µmoles of TCE to ethene every week. For each experiment the T3MP1 culture was purged with N2/CO2 (80:20 vol%) to remove trace chlorinated compounds and left to starve for four days to remove mRNA. Subsequently, 1.2 L of culture was split into two equal treatments of 600 mL in 1L bottles (Pyrex, VWR, Mississauga, ON) in an anaerobic glovebox (Coy Laboratory products, Grass Lake, MI). Into one treatment 5 mL (200 µmoles) of vinyl chloride was added as the electron acceptor and 15 µL (370 µmoles) methanol as the electron donor (VC+MeOH treatment). In the other treatment only the electron donor methanol was added (MeOH only). For the first set of arrays, RNA was extracted from the MeOH only and VC+MeOH treatments after 3 hours when 35 µmoles of ethene had been produced in the VC+MeOH treatment. A second set of experiments was performed a month later with the same set-up. For these, RNA was extracted after 3.5 hours when 50 µmoles of ethene had been produced in the VC+MeOH treatment. Within each biological set there were dye-switched duplicates, and technical replicates resulting in 6 arrays comparing the same treatments.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, 50 ml samples were withdrawn from the culture bottles inside an anaerobic chamber. After lysis with SDS and bead beating RNA was extracted using an ice-cold acid-phenol:chloroform:isoamyl alcohol method(Waller et al., 2005). The RNA solution was further purified using an RNeasy spin column (Qiagen,Valencia, California), and contaminating DNA was removed using the DNA-free kit (Ambion, Austin, TX). The quantity of the RNA was assessed using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the quality was assessed by electrophoresis on a 1% agarose gel or using the 2100 Bioanalyzer (Agilent Technologies).
Label
Cy5
Label protocol
The RNA was labelled using an indirect method in which amino allyl-dUTPs were incorporated during reverse transcription (RT), and then monoreactive Cyanine dyes were coupled to the aminoallyl cDNA in a separate reaction. First strand cDNA synthesis was carried out using 10-20 μg of total RNA, and 10 ng control RNA (Arabadopsis chlorophyll synthetase gene). The Cy3 or Cy5 labelled cDNA was purified then quantified using the NanoDrop ND-1000 to ensure that an appropriate amount of dye was incorporated (>100 pmoles) at an appropriate frequency (25-50 nucleotides per dye molecules). The labeled-cDNA was then hybridized to the slide at 37 0 C for 16-18 hours. The slides were then washed, dried and scanned with an Axon scanner using GenePix Pro software.
Hybridization protocol
The cDNA was then combined with 80 μL of hybridization solution (80 μL DIG Easy Hyb (Roche, Indianapolis, IN), 4 uL calf thymus DNA (SIGMA, 10 mg/mL) and 4 uL yeast tRNA (Invitrogen, 10 mg/mL). After mixing the hybridization mixture was heated up to 65 0 C for 3 minutes and then allowed to cool to room temperature before being applied to the slide. The slide was then place in a hybridization chamber and placed in a 37 0 C incubator for 16-18 hours.
Scan protocol
After removing the coverslip in 1X SSC, two 15 minute washes were carried out at 55 0 C in 1XSSC/0.1%SDS each with intermittent agitation. Then an additional wash was performed in 0.1XSSC/0.1%SDS. The slides were then rinsed twice at room temperature in 0.1XSSC. The slides were subsequently dried by centrifugation at 600 rpm for 5 minutes in a slide box lined with Whatman paper and then immediately scanned with an Axon scanner using GenePix Pro software
Description
19,000 spot Custom shotgun metagenome microarrays constructed from short insert gDNA libraries of dechlorinating microbial community KB1.
Data processing
Data analysis was performed in the R programming language using programs available from Bioconductor such as Linear Analysis of Microarrays (Limma), gplots and other graphing programs. The mean values from each channel were log2 transformed and normalizedwithin each array using the LOWESS algorithm to remove intensity dependent effects. Normalised values were used to calculate the Cy3/Cy5 fluorescence ratios for each slide. Cy3/Cy5 ratios were then normalized between arrays using the 'scale' method that log ratios were scaled to have the same median-absolute-deviation across the arrays . Then linear modell to average, and bayesian to assess statistical significance. and biological repeats (seperate chemostat runs)with all replicates combined. Data was compiled from two biological repelicates, totalling six replicates all comparing VC+MeOH to MeOH-only.
