Originally purchased from American Type Culture Collection (Manassas, VA, USA).
Treatment protocol
This bottle was challenged with a sublethal amount of butanol (50 mM, 0.46% v/v). This sample was taken 60 minutes after the challenge.
Growth protocol
C. acetobutylicum ATCC 824 (American Type Culture Collection, Manassas, VA) was stored at -85 degree C in clostridial growth media (CGM) (94) containing 15% glycerol. Solid cultures were carried out on agar-solidified 2xYTG (YTG is 8 g/liter tryptone, 5 g/liter yeast extract, 2 g/liter NaCl, 2.5 g/liter glucose, and 7.5 g/l agar, pH 5.8) plates. Liquid cultures were inoculated with single colonies from plates at least 4 days old and heat shocked at 70 to 80°C for 10 min. Cultures were grown in an anaerobic chamber (Forma Scientific, Marietta, OH) in Clostridial Growth Medium (CGM, Wiesenborn, D. P., et al. 1988. Appl. Environ. Microbiol. 54:2717-2722) supplemented with 80 g/liter glucose until they reached an A600 ca. 0.6 (corresponding to midexponential growth). The cultures were then split equally into two bottles, one was stressed with butyrate and the other was used as control. Both were sampled at the same time points through the length of the experiment.
Extracted molecule
total RNA
Extraction protocol
Cell pellets were collected by centrifuging 3 to 10 mL of cultures at 5,000xg for 10 min, 4 degrees C. Cells were then lysed by resuspending in 200 microL SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0]) with 20 mg/mL lysozyme (Sigma) and incubation at 37 degrees C for 4 min. One milliliter of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added and the sample was vortexed. Samples were immediately stored at -85 degrees C and used within one month to minimize RNA degradation. To isolate and purify the RNA, samples were thawed at room temperature; 500 microL of sample was diluted with an equal amount of ice cold TRIzol and then purified following manufacturer's instructions. The RNA was resuspended in 20-30 microL RNase-free water and quantitated in a spectrophotometer by measuring absorbance at 260 and 280 nm. Samples were only used if the A260/A280 ratio was greater than 1.8. RNA integrity was also verified by running 0.5 microL of sample on a 1.0% agarose gel. Purified RNA were stored at -85 degree C and used within one week of purification.
Label
Cy3
Label protocol
An indirect labeling approach was followed where cDNA was first made in the presence of nucleotides and aminoallyl dUTP, purified, and then the aminoallyl dUTP groups were coupled to a monoreactive dye (Cy3 or Cy5). Total RNA (25 microg) was mixed with 15 microg random hexamers (Roche, Indianapolis, IN, USA) and the volume brought up to 18.5 microL with RNase-free water. Samples were incubated at 70 degrees C for 10 min and then snap-frozen in a dry ice/ethanol bath for 30 sec. Deoxynucleotide triphosphates (Invitrogen; 0.6 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, and 0.36 mM dTTP), 0.24 mM aminoallyl dUTP (Sigma), 400 units SuperScript II reverse transcriptase (Invitrogen), First Strand Buffer (Invitrogen), and 6 microM dithiothreitol were added to a final volume of 30 microL. The samples were incubated for 3 h to overnight at 42°C, and the reaction was stopped by addition of 0.5 M EDTA (pH 8.0). The remaining RNA was degraded by addition of NaOH (final concentration 11 mM), incubation at 65oC for 15 min, and neutralization with HCl (final concentration 11 mM). The cDNA was purified by bringing up the volume to 450 microL with sterile water and concentrating three times on a Millipore YM-30 column. The samples were dried in a rotary SpeedVac, redissolved in 4.5 microL of a 0.1 M sodium carbonate buffer (pH 9.0), and combined with 54 nmol NHS-ester Cy3 or Cy5 monofunctional dye (Amersham Biosciences) dissolved in an equal volume of DMSO. The mixture was incubated in the dark for 1 h. To purify the labeled cDNA, 35 microL of a 100 mM sodium acetate solution (pH 5.2) was added before purifying on a Qiagen Qiaquick PCR purification column (Valencia, CA, USA). cDNA quantity, quality, and dye incorporation was measured (A260, A280, A550 [Cy3 probe], and A649 [Cy5 probe]) on a spectrophotometer. Only samples with a dye incorporation of at least 10 dye molecules per 1,000 nucleotides were hybridized on microarrays. Samples were stored at -20 degrees C for up to 3 weeks until microarray hybridization.
Originally purchased from American Type Culture Collection (Manassas, VA, USA).
Treatment protocol
This bottle was not subject to any challenge and it is used as a control. This sample was taken 60 minutes after the challenge.
Growth protocol
C. acetobutylicum ATCC 824 (American Type Culture Collection, Manassas, VA) was stored at -85 degree C in clostridial growth media (CGM) (94) containing 15% glycerol. Solid cultures were carried out on agar-solidified 2xYTG (YTG is 8 g/liter tryptone, 5 g/liter yeast extract, 2 g/liter NaCl, 2.5 g/liter glucose, and 7.5 g/l agar, pH 5.8) plates. Liquid cultures were inoculated with single colonies from plates at least 4 days old and heat shocked at 70 to 80°C for 10 min. Cultures were grown in an anaerobic chamber (Forma Scientific, Marietta, OH) in Clostridial Growth Medium (CGM, Wiesenborn, D. P., et al. 1988. Appl. Environ. Microbiol. 54:2717-2722) supplemented with 80 g/liter glucose until they reached an A600 ca. 0.6 (corresponding to midexponential growth). The cultures were then split equally into two bottles, one was stressed with butyrate and the other was used as control. Both were sampled at the same time points through the length of the experiment.
Extracted molecule
total RNA
Extraction protocol
Cell pellets were collected by centrifuging 3 to 10 mL of cultures at 5,000xg for 10 min, 4 degrees C. Cells were then lysed by resuspending in 200 microL SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0]) with 20 mg/mL lysozyme (Sigma) and incubation at 37 degrees C for 4 min. One milliliter of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added and the sample was vortexed. Samples were immediately stored at -85 degrees C and used within one month to minimize RNA degradation. To isolate and purify the RNA, samples were thawed at room temperature; 500 microL of sample was diluted with an equal amount of ice cold TRIzol and then purified following manufacturer's instructions. The RNA was resuspended in 20-30 microL RNase-free water and quantitated in a spectrophotometer by measuring absorbance at 260 and 280 nm. Samples were only used if the A260/A280 ratio was greater than 1.8. RNA integrity was also verified by running 0.5 microL of sample on a 1.0% agarose gel. Purified RNA were stored at -85 degree C and used within one week of purification.
Label
Cy5
Label protocol
An indirect labeling approach was followed where cDNA was first made in the presence of nucleotides and aminoallyl dUTP, purified, and then the aminoallyl dUTP groups were coupled to a monoreactive dye (Cy3 or Cy5). Total RNA (25 microg) was mixed with 15 microg random hexamers (Roche, Indianapolis, IN, USA) and the volume brought up to 18.5 microL with RNase-free water. Samples were incubated at 70 degrees C for 10 min and then snap-frozen in a dry ice/ethanol bath for 30 sec. Deoxynucleotide triphosphates (Invitrogen; 0.6 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, and 0.36 mM dTTP), 0.24 mM aminoallyl dUTP (Sigma), 400 units SuperScript II reverse transcriptase (Invitrogen), First Strand Buffer (Invitrogen), and 6 microM dithiothreitol were added to a final volume of 30 microL. The samples were incubated for 3 h to overnight at 42°C, and the reaction was stopped by addition of 0.5 M EDTA (pH 8.0). The remaining RNA was degraded by addition of NaOH (final concentration 11 mM), incubation at 65oC for 15 min, and neutralization with HCl (final concentration 11 mM). The cDNA was purified by bringing up the volume to 450 microL with sterile water and concentrating three times on a Millipore YM-30 column. The samples were dried in a rotary SpeedVac, redissolved in 4.5 microL of a 0.1 M sodium carbonate buffer (pH 9.0), and combined with 54 nmol NHS-ester Cy3 or Cy5 monofunctional dye (Amersham Biosciences) dissolved in an equal volume of DMSO. The mixture was incubated in the dark for 1 h. To purify the labeled cDNA, 35 microL of a 100 mM sodium acetate solution (pH 5.2) was added before purifying on a Qiagen Qiaquick PCR purification column (Valencia, CA, USA). cDNA quantity, quality, and dye incorporation was measured (A260, A280, A550 [Cy3 probe], and A649 [Cy5 probe]) on a spectrophotometer. Only samples with a dye incorporation of at least 10 dye molecules per 1,000 nucleotides were hybridized on microarrays. Samples were stored at -20 degrees C for up to 3 weeks until microarray hybridization.
Hybridization protocol
Oppositely labeled microarray probe pairs (5 microg of each) were mixed together, dried in a rotary SpeedVac, and redissolved in Pronto Universal Hybridization Solution. The samples were incubated at 95 degrees C for 5 min and loaded on the microarray under a LifterSlip (Erie Scientific, Portsmouth, NH, USA). The microarrays were placed in a Corning hybridization chamber with 50 microL 10xSSC (1xSSC is 0.15 M NaCl and 0.015 M sodium citrate) to maintain humidity. Microarrays were hybridized for 14-16 h in a 42 degrees C water bath. Following hybridization, microarrays were washed in TeleChem (Sunnyvale, CA, USA) wash buffers A (42 degrees C, 5 min), B (room temperature, 5 min), and C (room temperature, 2 min) and water. Slides were then spun dry at 240xg for 10 min.
Scan protocol
Scanned using a G2565BA Agilent Microarray Scanner (Agilent, Wilmington, DE, USA) at 10 microm resolution.
GenePix software (Molecular Devices, Union City, CA, USA) (v. 5.x) was used to assess spot quality and extract feature intensity statistics; duplicate spots were averaged; background-subtracted data were normalized using SNNLERM (Yang et al., PNAS 2003; 100:1122-1127).
