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| Status |
Public on Jun 02, 2009 |
| Title |
Clostridium beijerinckii mutant BA101 time course (13hr fermentation) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Clostridium beijerinckii NCIMB 8052 mixture of RNA samples collected at various time points
|
| Organism |
Clostridium beijerinckii NCIMB 8052 |
| Characteristics |
A pool of samples representating different stages of fermentation of Clostridium beijerinckii NCIMB 8052
|
| Biomaterial provider |
Blaschek Laboratory Stock
|
| Treatment protocol |
Samples were taken at different stages of fermentation from a batch culture of Clostridium beijerinckii NCIMB 8052 in Complete P2 Medium. Samples were combined to produce a pool for the generation of a reference probe for array hybridization.
|
| Growth protocol |
Clostridium beijerinckii NCIMB 8052 spore stock was stored at 4 oC in sterile H2O. Solid culture was prepared in agar-solidified TGY plates (TGY containing 30 g/L tryptone, 20 g/L glucose, 10 g/L yeast extract, 1 g/L L-cysteine and 4.5 g/L agar). Liquid cultures were inoculated with single colonies from plates. Cultures were grown in Cooked Meat Medium (CMM containing 100 g/L cooked meat medium and 10 g/L glucose) to late exponential phase at 35 °C in an anaerobic chamber. CMM culture was then inoculated into fresh TGY medium and grown at 35 °C for a few hours. TGY pre-culture was inoculated into Complete P2 Medium containing P2 stock solutions supplemented with 60 g/L glucose and 1 g/L yeast extract (P2 Medium, Qureshi, N. and Blaschek, H. P. 1999. Biomass Bioenergy 17:175-184) and grown at 35 °C into late stationary phase. Samples were taken at selected time points during the course of fermentation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 ml sample was collected at a specific time point from a batch fermentation culture. Cells were pelleted by centrifuging at 4,000 x g at 4 oC for 10 min. Total RNA was extracted from cell pellets using RNeasy Mini Kits (Qiagen) according to the manufacturer’s protocol. RNA quality was determined using nanochip on an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA concentration was quantified by A260 using a UV/vis spectrophotometer (Biotek Instruments). A RNA pool was generated by mixing equal amounts of RNA preparations obtained from samples taken at different time points during fermentation.
|
| Label |
Cy5
|
| Label protocol |
3ug total RNA was spiked with 6 ug random hexamer primers (GE Healthcare) in a 20 ul reaction, incubated at 70 oC for 10 min and then cooled on ice. The spiked reaction was incubated with 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, 0.2 mM aminoallyl-dUTP and 400 units Superscript III reverse transcriptase (Invitrogen) in 1X First Strand Buffer (Invitrogen) in a total of 40 ul at 46 oC for 3-8 hr. Aminoallyl-labelled cDNA in the mixture was purified on a QIAquick PCR purification column (QIAGEN) and dried in a speedvac. Cy3 or Cy5 dye ester (Molecular Probes) was dissolved in 10 ul 100 mM Na2CO3 and mixed with dried cDNA sample. The dye-coupling reaction was incubated in the dark at room temperature for 1 hr. The reaction was neutralized with 395 ul of a 300 mM sodium acetate solution (pH 5.2). The mixture was loaded onto a QIAquick PCR purification column (QIAGEN) to purifiy dye-labelled cDNA. The quantity, quality and dye incorporation ratio of purified cDNA probes were determined by measuring A260, A280, A550 (for Cy3 probe) and A650 (for Cy5 probe) on a UV/vis-spectrophotometer. Probes with dye incorporation of at least 20 dyes per 1,000 nucleotides were used for array hybridization. Purified cDNAs were dried on a speedvac and used for hybridization within the same day.
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| |
|
| Channel 2 |
| Source name |
Clostridium beijerinckii mutant BA101 13hr fermentaton sample
|
| Organism |
Clostridium beijerinckii |
| Characteristics |
Sample collected 13 hours after fermentor inoculation
|
| Biomaterial provider |
Blaschek Laboratory Stock
|
| Treatment protocol |
A Sample was taken at 13hr after initiation of a batch fermentation culture of Clostridium beijerinckii BA101 in Complete P2 Medium.
|
| Growth protocol |
Same as Channel 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Same as Channel 1 except that total RNA was extracted from 13hr fermentation culture
|
| Label |
Cy3
|
| Label protocol |
Same as Channel 1
|
| |
|
| |
| Hybridization protocol |
The printed array slides were re-hydrated and UV cross-linked. The slides were then washed in 0.2% SDS (w/v) followed by a H2O rinse. Washed slides were pre-hybridized in 75 mM sodium citrate and 750 mM NaCl pH 7.0 (5X SSC), 0.1% SDS and 1% (w/v) bovine serum albumin (BSA) in a 42 oC water bath for 45 min. After pre-hybridization the slides were washed in H2O five times followed by a last wash in isopropanol. The slides were spun dry immediately in 50 ml conical tubes at 700 x g for 1 min and kept until hybridization. A set of oppositely labeled cDNA probes were mixed in 30 ul SlideHyb Glass Array Hybridization Buffer (Ambion) and heated at 95 oC for 2-3 min. The probe set solution was applied to an array slide placed in a Corning hybridization chamber. The slides were hybridized in a 42 oC water bath for 16-20 hr. After hybridization the slides were washed with 1X SSC and 0.2% SDS at 42 oC for 5 min, followed by a second wash in 0.1X SSC and 0.2% SDS at room temperature for 5 min and two washes in 0.1X SSC at room temperature for 5 min. The washed slides were spun dry immediately at 200 x g for 5 min.
|
| Scan protocol |
Slides were scanned using an Axon 4000B Microarray Scanner (Molecular Devices).
|
| Description |
Clostridium beijerinckii mutant BA101 was monitored from exponential growth to stationary phase.
|
| Data processing |
Feature intensity statistics were extracted using GenePix Pro 6.0 (Molecular Devices). Median spot intensity was obtained by substracting median intensity of non-specific background from median intensity of each feature. Spot intensities for all the features on an array were normalized using TIGR MIDAS. Expression ratios for duplicate spots representing the same probe sequence were averaged after normalization.
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| Submission date |
Aug 06, 2008 |
| Last update date |
Jun 02, 2009 |
| Contact name |
Zhen Shi |
| E-mail(s) |
zshi@illinois.edu
|
| Organization name |
University of Illinois
|
| Department |
Institute for Genomic Biology
|
| Street address |
1206 West Gregory Drive
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
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| Platform ID |
GPL6990 |
| Series (2) |
| GSE12359 |
Clostridium beijerinckii BA101 mutant fermentation time course |
| GSE12365 |
Clostridium beijerinckii fermentation time course |
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