|
| Status |
Public on Feb 28, 2011 |
| Title |
Clostridium acetobutylicum ATCC 824 SigE mutant vs. SigF mutant (Hour 24) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. acetobutylicum SigF mutant, 24 hrs
|
| Organism |
Clostridium acetobutylicum ATCC 824 |
| Characteristics |
genotype: SigF mutant
timepoint: 24 hrs
|
| Growth protocol |
Colonies were grown on solid 2xYTG plates at 37°C under anaerobic conditions. Mutants were grown on or in media containing 5 µg/ml thiamphenicol. Individual colonies were picked and put in 10 ml of acetate-buffered CGM. WT colonies more than 5 days old were heat-shocked for 10 min at 70-80°C. Mutant colonies were not heat-shocked and were less than 3 days old. Tubes were grown to an OD600 ~1.0 and then used to inoculate 400 ml of acetate-buffered CGM (2.5% inoculum). Flasks were allowed to grow for 24 hrs before inoculating BioFlo 310 Benchtop Fermentors (10% inoculum). Fermentors were operated as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.]. Glucose levels were kept above 200 mM by adding 3.5 M glucose as needed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.].
|
| Label |
Cy3,Cy5
|
| Label protocol |
cDNA was generated and labeled as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.].
|
| |
|
| Channel 2 |
| Source name |
C. acetobutylicum SigE mutant, 24 hrs
|
| Organism |
Clostridium acetobutylicum ATCC 824 |
| Characteristics |
timepoint: 24 hrs
genotype: SigE mutant
|
| Growth protocol |
Colonies were grown on solid 2xYTG plates at 37°C under anaerobic conditions. Mutants were grown on or in media containing 5 µg/ml thiamphenicol. Individual colonies were picked and put in 10 ml of acetate-buffered CGM. WT colonies more than 5 days old were heat-shocked for 10 min at 70-80°C. Mutant colonies were not heat-shocked and were less than 3 days old. Tubes were grown to an OD600 ~1.0 and then used to inoculate 400 ml of acetate-buffered CGM (2.5% inoculum). Flasks were allowed to grow for 24 hrs before inoculating BioFlo 310 Benchtop Fermentors (10% inoculum). Fermentors were operated as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.]. Glucose levels were kept above 200 mM by adding 3.5 M glucose as needed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.].
|
| Label |
Cy5,Cy3
|
| Label protocol |
cDNA was generated and labeled as described in [Jones SW, et al. Genome Biol. 2008;9(7):R114.].
|
| |
|
| |
| Hybridization protocol |
Microarrays were hybridized and washed according to Agilent Technologies' recommended protocols.
|
| Scan protocol |
Microarrays were scanned according to Agilent Technologies' recommended protocols.
|
| Data processing |
Data was normalized using the LOESS method and implemented using the Bioconductor package in the statistical application R [Dudoit S, et al. Bioconductor R packages for exploratory analysis and normalization of cDNA microarray data. The Analysis of Gene Expression Data: Methods and Software. Springer, NY, 2002.][Yang YH, et al. Nucleic Acids Res. 2002;30(4):e15.]. Dye swaps and replicate spots were averaged together to give one expression value per gene. If the spot's intensity was not greater than 50, the spot was assigned a value of NA because it was not above background intensity [Jones SW, et al. Genome Biol. 2008;9(7):R114.].
|
| |
|
| Submission date |
Sep 13, 2010 |
| Last update date |
Feb 28, 2011 |
| Contact name |
Eleftherios Terry Papoutsakis |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Delaware
|
| Department |
Chemical Engineering
|
| Street address |
15 Innovation Way
|
| City |
Newark |
| State/province |
DE |
| ZIP/Postal code |
19711 |
| Country |
USA |
| |
|
| Platform ID |
GPL10908 |
| Series (1) |
| GSE24103 |
Regulon prediction of the sporulation factors Spo0A, SigF, SigE, and SigG in Clostridium acetobutylicum ATCC 824 |
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