|
| Status |
Public on May 23, 2007 |
| Title |
E.coli_BCadapted3_replicate3_50ppmBC |
| Sample type |
RNA |
| |
|
| Source Name |
E. coli adapted in benzalkonium chloride
|
| Organism(s) |
Escherichia coli |
| Characteristics |
E. coli K-12 ATCC47076 adapted to grow in benzalkonium chloride and pregrown in 50 ppm benzalkonium chloride
|
| Biomaterial provider |
American Type Culture Collection
|
| Treatment protocol |
Frozen cultures were inoculated in 5 ml TSB and incubated over-night. Two drops culture was inoculated in 5 ml TSB with benzalkonium chloride (BC) in a range of concentrations in 2 µg ml-1 steps. The cultures were incubated over night (20-24 h) and inspected visually for growth. The tube with highest BC concentration with visible growth was reinoculated (two drops) in tubes with fresh TSB, with a range of BC-concentrations in steps of 2 µg ml-1. The cultures were incubated over-night and inspected visually for growth. This procedure was continued in a total of 30-40 days until five reinoculations did not result in growth at higher concentrations of BC.
|
| Growth protocol |
Frozen cultures were inoculated in 5 ml TSB and incubated over-night. One ml culture was inoculated in 99 ml TSB with 50 µg ml-1 benzalkonium chloride in 500 ml baffled shake flask and incubated at 37 oC with shaking. Bacterial cells were harvested at mid log phase (~108 CFU ml-1).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cells were harvested directly from shake flask into RNAprotect (Qiagen) to secure stable RNA before cell lysis. One volume of culture (1x108 CFU ml-1) was added to two volumes of RNA protect, vortexed and incubated at room temperature for 5 min. The sample was then centrifuged at 5000 g for 10 min and the supernatant removed.Total RNA was isolated using RNeasy mini kit (Qiagen) according to the manufacturer’s recommendations. The concentration of RNA was determined by measuring the absorbance at 260 nm and 280 nm in an Ultrospec 3000 spectrophotometer (Pharmacia Biotech). The ratio between the measured absorbance at the two wavelengths shows an estimate of RNA purity with respect to contamination. (Qiagen Bench guide). RNA degradation was checked with an Agilent 2100 Bioanalyzer according to userguide from Agilent Technologies. Before cDNA labelling, variations in RNA concentrations were adjusted to 1 µg µl-1 for all array hybridizations.
|
| Label |
33P
|
| Label protocol |
Labeled cDNA was prepared according to the PanoramaTM E. coli gene array protocol (Sigma Genosys), with some modifications. SuperscriptTM II Rnase H- Reverse Transcriptase (Invitrogen) and Sheared Salmon Sperm DNA (Ambidon) replaced equivalent components given in the Panorama protocol. The unincorporated nucleotides were removed with MicroSpinTM G-25 columns (Amersham Pharmacia Biotech), according to given protocol.
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| |
|
| Hybridization protocol |
Purified labeled cDNA were hybridised to the PanoramaTM E. coli gene array, according to supplied protocol. [33P] dCTP from Amersham Bioscience was used for all hybridisations.
|
| Scan protocol |
The membranes were wrapped in clear plastic film and exposed to a Storage Phosphore Screen (Amersham Biosciences) for 6-7 days. After exposure the screens were scanned with Thyphoon 8600 variable mode imager (Amersham Biosciences).
|
| Description |
The arrays were stripped between hybridisations according to the PanoramaTM E. coli gene arrays technical protocol (Sigma Genosys).
|
| Data processing |
The software Imagene (BioDiscovery) was used for quantification of data and the software Genespring (Silicon Genetics) for normalisation. Pixel density for each spot was determined by laying down a grid of individual circles corresponding to each spot and the grid template for E. coli K-12 (Sigma genosys) was loaded for gene identification of each spot. No genes were flagged or no cut-off were made before normalisation in GeneSpring. The data was normalised by subtracting background based on all empty spots (negative controls) on the array. In addition each array was normalised to 50th percentile to adjust signal intensity of each array to all arrays. Each array was defined as independent samples in further data analysis.
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| |
|
| Submission date |
Jan 11, 2007 |
| Contact name |
Solveig Langsrud |
| Organization name |
Matforsk, Norwegian Food Research Institute
|
| Street address |
Osloveien 1
|
| City |
Aas |
| ZIP/Postal code |
1430 |
| Country |
Norway |
| |
|
| Platform ID |
GPL189 |
| Series (1) |
| GSE6712 |
Adapted tolerance to Benzalkonium chloride in E. coli K12 |
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