|
| Status |
Public on Apr 14, 2008 |
| Title |
E.coli/Hsp60-wt/wt(-/+) |
| Sample type |
RNA |
| |
|
| Source name |
E. coli B178 groESL(-) expressing human Hsp60 and Hsp10
|
| Organism |
Escherichia coli |
| Characteristics |
E. coli strain B178 (Georgopoulos et al. (1973) J Mol Biol 76: 45-60) in which the endogenous groES and groEL genes have been deleted and that is maintained alive by the pOFX-tac1-derived plasmid carrying an IPTG-inducible operon with cDNA encoding human Hsp10 and the mature part of human wild type Hsp60 (Hansen et al., 2002). These cells were subsequently transformed by electroporation with a second plasmid, derivative of pBAD/hisA (Invitrogen), that contained an operon with Hsp10 and wild type Hsp60 under control of the arabinose-inducible BAD promoter. The pBAD/hisA-derived plasmids carry a pBR322 origin of replication and an ampicillin resistance gene and can be stably maintained in the same cell with the p15A-origin/kanamycin resistance gene containing pOFX-tac derivative.
|
| Growth protocol |
Bacterial cells were grown in dYT medium (Miller, JH (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) supplemented with kanamycin (10 mg/L), ampicillin (100 mg/L) and IPTG (0.1mM) at 30°C in a shaking water bath. At a turbidity (measured at OD536) of approximately 0.1 bacteria were harvested by centrifugation at ambient temperature and resuspended in fresh dYT medium containing antibiotics and 0.2% arabinose. Growth was continued at 30°C and 10 hours after inducer shift 3ml sample were withdrawn, treated with RNAPROTECT (Qiagen, Ballerup), harvested by centrifugation and frozen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using the RNAeasy kit (Qiagen).
|
| Label |
phycoerythrin
|
| Label protocol |
Affymetrix standard
|
| |
|
| Hybridization protocol |
Affymetrix standard
|
| Scan protocol |
Affymetrix standard
|
| Description |
Isolated RNA from 2 parallel cultures originating from 2 individual transformants was pooled and used for cDNA synthesis and labelling.
|
| Data processing |
MAS5.0
|
| |
|
| Submission date |
Mar 28, 2008 |
| Last update date |
Apr 14, 2008 |
| Contact name |
Peter Bross |
| E-mail(s) |
peter.bross@ki.au.dk
|
| Phone |
+4589495147
|
| Fax |
+4589496018
|
| Organization name |
Aarhus University Hospital, Skejby
|
| Department |
Institute for Clinical Medicine
|
| Lab |
Research Unit for Molecular Medicine
|
| Street address |
Brendstrupgaardsvej 100
|
| City |
Århus |
| ZIP/Postal code |
8200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL199 |
| Series (1) |
| GSE10974 |
Expression profiles of E. coli groESL(-) cells expressing human Hsp60(wt)/Hsp10 or Hsp60-(p.Val98Ile)/Hsp10 operons |
|
