|
| Status |
Public on May 18, 2007 |
| Title |
pGIT1.1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pGIT1
|
| Organism |
Escherichia coli |
| Characteristics |
Strain: MG1655 pGIT1 (carries the ytfE promoter in multicopy, titrates out NsrR to phenocopy an NsrR- mutation) Growth: Anaerobic to early exponential growth Media:Minimal salts + 20 mM trimethylamine-N-oxide
|
| Biomaterial provider |
Lesley Griffiths, J.A.Cole laboratory
|
| Treatment protocol |
15 ml of culture was mixed with 30 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy midikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy5
|
| Label protocol |
20 ug total RNA was converted to Cy5-labelled cDNA
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy5 NHS-dye esters to the amino-allyl-labelled
cDNAs
|
| |
|
| Channel 2 |
| Source name |
pGIT8
|
| Organism |
Escherichia coli |
| Characteristics |
Strain: MG1655 pGIT8 (carries 1 base pair deletion in the NsrR-binding site of the ytfE promoter) Growth: Anaerobic to early exponential growth Media:Minimal salts + 20 mM trimethylamine-N-oxide Pool of eight independent cultures
|
| Biomaterial provider |
Lesley Griffiths, J.A.Cole laboratory
|
| Treatment protocol |
15 ml of culture was mixed with 30 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy midikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy3
|
| Label protocol |
20 ug total RNA was converted to Cy3-labelled cDNA
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy3 NHS-dye esters to the amino-allyl-labelled
cDNAs
|
| |
|
| |
| Hybridization protocol |
solution at 42 °C and washed twice in 0.1xSSC, 0.1% SDS for 30 s at room temperature. Slides were immediately transferred into prewarmed prehybridization solution (5xSSC, 0.1% SDS, and 0.1% bovine serum albumin) and incubated for 2–4 h at 42 °C. The microarray slides were finally washed at room temperature once in 0.1xSSC, 0.1% SDS for 1 min and twice in 0.1xSSC for 30 s, briefly dipped in water and then ethanol, and dried by centrifugation for 5 min at 800xg. For each experiment, equal quantities (80 pmol) of each Cy5- and Cy3-labeled cDNA were added to a final volume of 80 microlitre of hybridization solution containing 25% formamide, 10 mg of bovine serum albumin (fraction V) per ml, 5xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 8 micrograms of poly(A), and 1xDenhardt’s solution.
|
| Scan protocol |
Slide was scanned at 532 and 630 nm by using a
Genepix 4000A scanner (Axon Instruments, Union City, CA).
|
| Description |
Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defence mechanisms, or as products of their own metabolism. The regulatory protein, NsrR (a member of the Rrf2 family of transcription factors), plays key roles in this stress response. Microarray analysis was utilised to reveal the full role of NsrR.
Regulation by NsrR is known to be extremely sensitive to repressor titration by NsrR-binding sites provided on a multicopy plasmid. Transformation of an nsrR+ strain with plasmid pGIT1, which carries the ytfE promoter in multicopy, titrates out NsrR to phenocopy an NsrR- mutation. As a control, pGIT8 carries the same promoter fragment, but a 1 bp deletion mutation in the NsrR binding site eliminates repressor titration in vivo. Strain MG1655 transformed with either pGIT1 was grown anaerobically and RNA was isolated from early exponential phase cultures grown in the absence of nitrite. A pool of RNA from the control strain (MG1655 transformed with pGIT8 and grown in the absence of nitrite) was included as a common reference.
|
| Data processing |
Data was analysed using GeneSpring
|
| |
|
| Submission date |
Jan 18, 2007 |
| Last update date |
Jan 25, 2007 |
| Contact name |
Chrystala Constantinidou |
| E-mail(s) |
[email protected]
|
| Phone |
+44 (0)121 414 5564
|
| Organization name |
University of Birmingham
|
| Department |
School of Biosciences
|
| Lab |
UBEC
|
| Street address |
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL3051 |
| Series (1) |
| GSE6781 |
The NsrR regulon of Escherichia coli K-12 |
|
