Methanocaldococcus jannaschii (DSM 2661) pressure: 7.8 atm temperature: 88C volume: 200 ml medium
Biomaterial provider
Boonchai Boonyaratanakornkit
Growth protocol
M. jannaschii was cultivated in a 1.15 L high pressure vessel (model GC-17; High Pressure Equipment Co.) at a temperature of 88C. An unbaffled glass liner was used to minimize cell contact with SS316. H2:CO2 (4:1 v/v) substrate was supplied into the head space of the reactor to bring the pressure to 7.8 atm. The system was operated with 200 ml of medium prepared as previously described (Park, CB and Clark, DS, Appl Environ Microbiol 68:3, 1458-1463).
Extracted molecule
total RNA
Extraction protocol
Samples were withdrawn from the high T-P bioreactor through a micrometer control valve (High Pressure Equipment, Co.), passed through a 0.45 um nitrocellulose filter (Millipore) to collect cells. This filter was placed in cold phenol with 1% SDS and 0.1 M sodium acetate and homogenized using a beadbeater (BioSpec Products, Inc.) for 5 min at the highest setting. The aqueous layer was removed and re-extracted with cold phenol. Total RNA was precipitated overnight at -20C with 3M sodium acetate and isopropanol, treated with DNase (Invitrogen), and cleaned with RNeasy columns (Qiagen).
Label
Cy3
Label protocol
Two ug of total RNA and 6 ug of random hexamers (Invitrogen) were combined, incubated at 70C for 10 min, and snap frozen in a dry-ice/ethanol bath for 30 s. First-strand cDNA synthesis was carried out at 42C overnight with 400 U of SuperScript II (Invitrogen), 1X first-strand buffer (Invitrogen SuperScript II), 0.01 M DTT (Invitrogen SuperScript II), 12.5 mM dATP (Invitrogen), 12.5 mM dCTP (Invitrogen), 12.5 mM dGTP (Invitrogen), 4.16 mM dTTP (Invitrogen), and 8.33 mM aa-dUTP (Ambion). The reaction was quenched by adding 10 ul 0.5 M EDTA, and the RNA template was hydrolyzed with the addition of 10 ul 1 M NaOH followed by incubation at 65C for 15 min. The reaction was neutralized by adding 25 ul 1M Tris-HCl, pH 7.0 (Ambion). cDNA was separated from reaction reagents using a Microcon 30 spin column (Millipore) and lyophilized to dryness with a Speed Vac (Savant). For NHS-Cy dye coupling, sample was re-suspended in 4.5 ul 0.1 M sodium carbonate, pH 9.0, and 4.5 ul of either NHS-Cy3 or NHS-Cy5 (Amersham Pharmacia) and allowed to couple to the cDNA for 1 h at room temperature in the dark. The coupling reaction was quenched with 4.5 ul 4 M hydroxylamine and incubated in the dark for 15 min at room temperature. 35 ul 100 mM sodium acetate pH 5.2 was added and unincorporated dye was removed using QIAquick columns (Qiagen). The whole undiluted sample was analyzed using a spectrophotometer (Beckman) to determine dye incorporation and nucleotides per dye; targets with dye incorporation of over 10 pmol and nucleotides-per-dye ratios below 60 were used in hybridizations. The Cy3 and Cy5 labeled cDNA targets were lyophilized to dryness in a Speed Vac (Savant).
Channel 2
Source name
Methanocaldococcus jannaschii, 7.8 atm, 600 ml medium
Methanocaldococcus jannaschii (DSM 2661) pressure: 7.8 atm temperature: 88C volume: 600 ml medium
Biomaterial provider
Boonchai Boonyaratanakornkit
Growth protocol
M. jannaschii was cultivated in a 1.15 L high pressure vessel (model GC-17; High Pressure Equipment Co.) at a temperature of 88C. An unbaffled glass liner was used to minimize cell contact with SS316. H2:CO2 (4:1 v/v) substrate was supplied into the head space of the reactor to bring the pressure to 7.8 atm. The system was operated with 600 ml of medium prepared as previously described (Park, CB and Clark, DS, Appl Environ Microbiol 68:3, 1458-1463).
Extracted molecule
total RNA
Extraction protocol
Samples were withdrawn from the high T-P bioreactor through a micrometer control valve (High Pressure Equipment, Co.), passed through a 0.45 um nitrocellulose filter (Millipore) to collect cells. This filter was placed in cold phenol with 1% SDS and 0.1 M sodium acetate and homogenized using a beadbeater (BioSpec Products, Inc.) for 5 min at the highest setting. The aqueous layer was removed and re-extracted with cold phenol. Total RNA was precipitated overnight at -20C with 3M sodium acetate and isopropanol, treated with DNase (Invitrogen), and cleaned with RNeasy columns (Qiagen).
Label
Cy5
Label protocol
Two ug of total RNA and 6 ug of random hexamers (Invitrogen) were combined, incubated at 70C for 10 min, and snap frozen in a dry-ice/ethanol bath for 30 s. First-strand cDNA synthesis was carried out at 42C overnight with 400 U of SuperScript II (Invitrogen), 1X first-strand buffer (Invitrogen SuperScript II), 0.01 M DTT (Invitrogen SuperScript II), 12.5 mM dATP (Invitrogen), 12.5 mM dCTP (Invitrogen), 12.5 mM dGTP (Invitrogen), 4.16 mM dTTP (Invitrogen), and 8.33 mM aa-dUTP (Ambion). The reaction was quenched by adding 10 ul 0.5 M EDTA, and the RNA template was hydrolyzed with the addition of 10 ul 1 M NaOH followed by incubation at 65C for 15 min. The reaction was neutralized by adding 25 ul 1M Tris-HCl, pH 7.0 (Ambion). cDNA was separated from reaction reagents using a Microcon 30 spin column (Millipore) and lyophilized to dryness with a Speed Vac (Savant). For NHS-Cy dye coupling, sample was re-suspended in 4.5 ul 0.1 M sodium carbonate, pH 9.0, and 4.5 ul of either NHS-Cy3 or NHS-Cy5 (Amersham Pharmacia) and allowed to couple to the cDNA for 1 h at room temperature in the dark. The coupling reaction was quenched with 4.5 ul 4 M hydroxylamine and incubated in the dark for 15 min at room temperature. 35 ul 100 mM sodium acetate pH 5.2 was added and unincorporated dye was removed using QIAquick columns (Qiagen). The whole undiluted sample was analyzed using a spectrophotometer (Beckman) to determine dye incorporation and nucleotides per dye; targets with dye incorporation of over 10 pmol and nucleotides-per-dye ratios below 60 were used in hybridizations. The Cy3 and Cy5 labeled cDNA targets were lyophilized to dryness in a Speed Vac (Savant).
Hybridization protocol
Slides were pre-hybridized in filter-sterilized buffer (5X SSC, 0.1% SDS, 1% BSA) at 42C for at least 1 h. Slides were then washed in Milli-Q (Millipore) ultrapure H2O for 10 min, then in isopropanol for 6 min, and spun dry. Dried, labeled cDNA targets were resuspended in at least 10 ul of hybridization buffer (50% formamide, 5X SSC, 0.1% SDS, 1 ug/ul salmon sperm DNA) for 15 min at RT so that the dye concentrations in samples hybridized on the same slide were approximately the same. Cy5 and Cy3 labeled targets were combined, and targets were heated for 10 min at 95C and cooled on ice for 30 sec.
Scan protocol
Slides were scanned on a GenePix 4000 dual-color confocal laser scanner (Axon). Images were collected in the Cy3 and Cy5 channels (532 nm and 635 nm, respectively) and stored as paired TIFF images.
Description
Effect of gas substrate limitation on barophilic growth at 500 atm.
