|
| Status |
Public on Jan 26, 2012 |
| Title |
EGS212_MlfabH_rep_5 |
| Sample type |
RNA |
| |
|
| Source name |
EGS212
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
substrain: DH1
genotype: ∆fadE with overexpression of 'tesA and MlfabH via an IPTG-inudcible promoter
phenotype: small improvement in methyl ketone production
|
| Treatment protocol |
After 6 hours of growth both control and test strains were induced with 0.5mM IPTG and harvested 2 hours post-induction.
|
| Growth protocol |
Escherichia coli DH1 ∆fadE control and test strains were seeded with OD600nm 0.03 overnight cultures into 15ml tryptic soy broth media and grown at 37°C with 200rpm agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and the RNAprotect reagent (Qiagen) and DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). RNA concentration was determined on a Nanodrop ND-1000 (Thermo Scientific) and RNA quality was determined by analysis with an Agilent 2100 bioanalyzer.
|
| Label |
Alex fluor 555
|
| Label protocol |
Labeling was carried out by reverse transcription of 10 µg of total RNA primed with 5 µg of random hexamers (Roche, Germany) were reverse transcribed using the SuperScript Indirect cDNA labeling kit (Invitrogen). Alexa Fluor 555 dyes (Invitrogen) were then incorporated into amino-allyl-dUTP-labeled cDNA, the fluorescently labeled cDNA was purified with the QiaQuick PCR purification kit (Qiagen) and dried under vacuum (Vacufuge Speed Vac, Eppendorf).
|
| |
|
| Hybridization protocol |
Hybridization was performed at 42ºC for 20-24 hrs following NimbleGen's standard operating protocol. See www.nimblegen.com.
|
| Scan protocol |
Scanning was performed on a Axon GenePix 4200A scanner following NimbleGen's standard operating protocol. See www.nimblegen.com.
|
| Description |
This sample is of Escherichia coli DH1 delta-fadE with pKS1 and pEG205 plasmids. It is the fifth of five biological replicates of the test strain used in this experiment, each from separate cultures.
|
| Data processing |
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
|
| |
|
| Submission date |
Oct 03, 2011 |
| Last update date |
Jan 26, 2012 |
| Contact name |
Ee-Been Goh |
| E-mail(s) |
egoh@lbl.gov
|
| Organization name |
Joint BioEnergy Institute
|
| Department |
Fuels Synthesis Division
|
| Street address |
5885 Hollis St, 4th Floor
|
| City |
Emeryville |
| State/province |
CA |
| ZIP/Postal code |
94608 |
| Country |
USA |
| |
|
| Platform ID |
GPL14649 |
| Series (1) |
| GSE32561 |
Investigating the impact of heterologous expression of the Micrococcus luteus FabH on global transcription in Escherichia coli |
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