|
| Status |
Public on Mar 11, 2008 |
| Title |
E. coli AcrB and EmrAB Multidrug Efflux Double Mutant |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AcrB- and EmrAB-Negative, Multidrug Efflux Mutant Strain
|
| Organism |
Escherichia coli |
| Characteristics |
HNCE4 is an isogenic mutant derived from drug sensitive AG102MB (marR1, AcrB::kan). It contains an additional mutation at the emrAB locus (marR1, acrB::kan, emrAB::cat) resulting in a multidrug efflux negative phenotype.
|
| Growth protocol |
Cultures were grown to mid-log phase in Luria-Bertani (Miller) broth medium at 37C. Cells were cultured with 25 ug/ml chloramphenicol to maintain selection. However, this genomic mutation is stable and RNA was prepared from non-selectively grown cultures inoculated with overnight stocks (grown with selection) to eliminate any unrelated effects of exogenous drugs on gene expression.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen RNeasy kit.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was reverse transcribed with random primer mix and labeled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
Efflux Mutant Parent Strain
|
| Organism |
Escherichia coli |
| Characteristics |
The parent strain, AG102, was previously derived from AG100 (K-12). AG102 is drug hyper-resistant due to a mutation in marR (marR1) which increases expression of MarA, a global regulator, which in turn results in overexpression of the AcrAB-TolC multidrug efflux system.
|
| Growth protocol |
Cultures were grown to mid-log phase in Luria-Bertani (Miller) broth medium at 37C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen RNeasy kit.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was reverse transcribed with random primer mix and labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
These differentially labeled cDNAs were hybridized together on Corning Epoxide slides containing the Operon E. coli Genome Oligo Set Version 1.0 printed by the Microarray and Proteomics Facility, Department of Biological Sciences, University of Alberta. The hybridizations were performed in a hybridization cassette (ArrayIt) in a water bath at 45ºC for 18 hours. The slides were then washed in pre-warmed washing buffer 2X SSC containing 0.1% SDS for 5 minutes, 1X SSC for 5 minutes, and 0.5X SSC for 5 minutes.
|
| Scan protocol |
The spun-dried hybridized slides were scanned with a GenePix 4000B scanner (Axon Instruments) at 10 µm resolution using appropriate photomultiplier tube gains to obtain the highest intensity with <0.1% saturated pixels. The resulting images were analyzed by measuring the fluorescence of all features on the slides using the GenePix Pro 6.0 software (Axon Instruments). The median fluorescence intensity of these pixels within each feature was taken as the intensity value for the whole feature.
|
| Description |
A total of five independent microarray data sets were generated for the pairing (AG102MB X AG102) from five independent cultures and associated RNA preparations. Each microarray slide contains three full copies of the array design giving a total of 15 data points (5x3) for each unique spot.
|
| Data processing |
The analyzed data were imported to ArrayTrack (NCTR Center for Toxicoinformatics;http://www.fda.gov/nctr/science/centers/toxicoinformatics/ArrayTrack/arraytrack_webaccess.htm). The raw data were normalized by using median channel (intensity) scaling with no background subtraction.
|
| |
|
| Submission date |
Mar 07, 2008 |
| Last update date |
Mar 10, 2008 |
| Contact name |
Christopher A Elkins |
| E-mail(s) |
chris.elkins@fda.hhs.gov
|
| Phone |
870-543-7547
|
| Fax |
870-543-7307
|
| Organization name |
FDA National Center for Toxicological Research
|
| Department |
Microbiology
|
| Street address |
3900 NCTR Drive
|
| City |
Jefferson |
| State/province |
AR |
| ZIP/Postal code |
72079-9502 |
| Country |
USA |
| |
|
| Platform ID |
GPL6540 |
| Series (1) |
| GSE10757 |
Mutations in Multidrug Efflux Pumps of Eshcerichia coli |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM271754.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
| GSM271754_array1_bottom.gpr.gz |
612.9 Kb |
(ftp)(http) |
GPR |
| GSM271754_array1_middle.gpr.gz |
614.4 Kb |
(ftp)(http) |
GPR |
| GSM271754_array1_top.gpr.gz |
612.7 Kb |
(ftp)(http) |
GPR |
| GSM271754_array2_bottom.gpr.gz |
604.3 Kb |
(ftp)(http) |
GPR |
| GSM271754_array2_middle.gpr.gz |
615.8 Kb |
(ftp)(http) |
GPR |
| GSM271754_array2_top.gpr.gz |
606.5 Kb |
(ftp)(http) |
GPR |
| GSM271754_array3_bottom.gpr.gz |
614.6 Kb |
(ftp)(http) |
GPR |
| GSM271754_array3_middle.gpr.gz |
619.5 Kb |
(ftp)(http) |
GPR |
| GSM271754_array3_top.gpr.gz |
617.9 Kb |
(ftp)(http) |
GPR |
| GSM271754_array4_bottom.gpr.gz |
622.0 Kb |
(ftp)(http) |
GPR |
| GSM271754_array4_middle.gpr.gz |
628.4 Kb |
(ftp)(http) |
GPR |
| GSM271754_array4_top.gpr.gz |
622.2 Kb |
(ftp)(http) |
GPR |
| GSM271754_array5_bottom.gpr.gz |
637.2 Kb |
(ftp)(http) |
GPR |
| GSM271754_array5_middle.gpr.gz |
643.0 Kb |
(ftp)(http) |
GPR |
| GSM271754_array5_top.gpr.gz |
652.1 Kb |
(ftp)(http) |
GPR |
| Processed data included within Sample table |
