|
| Status |
Public on Jun 18, 2022 |
| Title |
thrL mutant grown in the absence of homoserine S3 |
| Sample type |
RNA |
| |
|
| Source name |
thrL mutant grown in the absence of homoserine
|
| Organism |
Escherichia coli |
| Characteristics |
genotype: {delta}thrL mutant
strain: E coli K12 -MG1655
treatment: absence of homoserine
|
| Treatment protocol |
the treatment was the cultivation of the E. coli strains in the absence or in the presence of 10 mM homoserine
|
| Growth protocol |
An overnight pre-culture of the different E.coli strains in M9 medium/1 % glucose was inoculated in 0.3 L shake flasks containing 50 ml of the same medium (initial OD600 of ∼0.2) in the absence or presence of 10 mM L-homoserine. Cultures were carried at 37 °C at 200 rpm until OD600 reached ~1.0 to 1.5. At this time, one ml of culture taken 3 to 5 times was collected in microcentrifuge tubes and centrifuged at 10,000g for 2 min. The cell pellets were quickly washed once with 1 ml cold water, resuspended and quickly centrifuged at as above. After draining remaining water on a towel, the tubes were thrown in liquid nitrogen and stored at -80°C until use. This procedure was repeated with three biological independent cultures and for each strain. RNA were extracted frozen cell pellets using the QIAGEN RNeasy Mini Kit RNA, quantified by NanoDrop (Thermo) and its quality control was validated on Bioanalyzer (Agilent Technologies). Only RNA samples with a RIN (RNA Integrity Number) higher than or equal to 9.00 were chosen for microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA were extracted frozen cell pellets using the QIAGEN RNeasy Mini Kit RNA, quantified by NanoDrop (Thermo) and its quality control was validated on Bioanalyzer (Agilent Technologies). Only RNA samples with a RIN (RNA Integrity Number) higher than or equal to 9.00 were chosen for microarray analysis.
|
| Label |
CY3
|
| Label protocol |
The RNA were converted to cDNA using the Low Input Quick Amp labeling kit (Agilent) with dCTP(CY3) labelling
|
| |
|
| Hybridization protocol |
The labeled cDNA was hybridized on E. coli Gene Expression Microarrays (8 × 15K, Agilent) following the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol
|
| Scan protocol |
The slides were scanned on a Tecan scanner MS200 and analyzed by Feature Extraction V.11.5.1.1
|
| Description |
DthrL_1_2
|
| Data processing |
Signals of each probe set were filtered according to the coefficient of variations with a cut-off value of 50 %. Significant changed expression was acquired by moderated t-test with a p-value of < 0.05 and a fold-change cut-off value of 2.0. Benjamini-Hochberg correction was performed (Hochberg and Benjamini, 1990).
Normalized signal intensity and log2 transformed in supplementary file.
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| |
|
| Submission date |
Jun 15, 2022 |
| Last update date |
Jun 18, 2022 |
| Contact name |
JEAN MARIE FRANCOIS |
| E-mail(s) |
fran_jm@insa-toulouse.fr
|
| Organization name |
Toulouse Biotechnology Institute
|
| Street address |
135 avenue de Rangueil
|
| City |
Toulouse |
| ZIP/Postal code |
31077 |
| Country |
France |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE206196 |
Transcriptomic analysis of homoserine-evolved MG1655 strain (wild type) and of homoserine -treated wild type, mutant deleted for thrL and mutant bearing thrL* allele |
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