|
| Status |
Public on Nov 24, 2010 |
| Title |
LKKI_42_4_1 |
| Sample type |
RNA |
| |
|
| Source name |
cell culture
|
| Organism |
Escherichia coli |
| Characteristics |
strain: production
growth temperature: 42
harvest od: 4
replicate: 1
|
| Treatment protocol |
After reaching suitable optical density at OD600nm ≈ 4 the inoculum was transferred to the GYSP medium and immediately induced with IPTG. The cultures were then incubated in shake flasks at 160 rpm and at three different temperatures (i.e. T = 25°C, 37°C and 42°C, respectively) until the appropriate culture´s optical density (OD), indicating the transition from the exponent to the stationary phase was reached (OD600nm ≈ 4 or 10 for the culture grown at 25 °C and OD600 nm ≈ 4 for the cultures grown at 37°C or 42°C). The cultivation experiment was repeated 3 times.
|
| Growth protocol |
Bacterial inoculum E. coli BL21 pET3a with inserted gene sequence coding for hG-CSF protein (production strain) or E. coli BL21 pET3a without inserted gene sequence coding for hG-CSF protein (control strain) was prepared in a shake flask culture and grown overnight at 25°C and at 160 rpm in the LBPG/amp100 medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were stabilized with RNA protect Bacteria Reagent (Qiagen), aliquoted, centrifuged and the bacterial pellet was stored at – 80°C for further. RNA isolation and DNase treatment was performed as described by Petek et al [BMC Mocrobiology, 2010], except for substituting lysostaphin with lysocyme (500 mg/ml) in the cell lysis step. RNA quality, quantity and integrity was checked by NanoDrop (NanoDrop Technologies, USA), gel electrophoresis and Bioanalyzer (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Purified RNA (approximately 30 µg) was used for the cDNA synthesis and direct labeling (Superscript II, Invitrogen). Luciferase control mRNA (1 ng/µg; Promega) and 3 µg of random primers were added to each RNA sample. This was followed by 10 minutes of incubation at 70°C and immediate chilling on ice. cDNA synthesis was carried out using SuperScript II reverse transcriptase (Invitrogen) according to manufacturer’s instructions. Synthesised cDNA was purified using MinElute PCR purification Kit (Qiagen). The concentration of cDNA, efficiency of dye (Cy5) integration and integrity of labeled cDNA were checked by NanoDrop and gel electrophoresis.
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| |
|
| Hybridization protocol |
Labeled cDNA was hybridized to the arrays according to the protocol recommended by CombiMatrix except for using 2X formamide based hybridization buffer (Genisphere) containing Salmon testis DNA (1 μg / μl, Sigma) and shorter hybridization time (1h).
|
| Scan protocol |
After hybridization semiconductor microarray surfaces were covered by imaging solution and were scanned using a fluorescence LS200 scanner (TECAN).
|
| Data processing |
Combimatrix Microarray Images Software was used for gridding, excluding bad quality spots and export of signal intensities. Further data analysis was peformed in R software environment for statistical computing and graphics (http://www.r-project.org/). Bioconductor´s package affy was used for quality control, preprocessing and statistical significance testing. Backround was not substracted. The data was normalized using the Quantile normalization.
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| |
|
| Submission date |
Nov 23, 2010 |
| Last update date |
Nov 23, 2010 |
| Contact name |
Spela Baebler |
| E-mail(s) |
spela.baebler@nib.si
|
| Organization name |
National Institute of Biology
|
| Department |
Department of Biotechnology and Systems Biology
|
| Street address |
Vecna pot 111
|
| City |
Ljubljana |
| ZIP/Postal code |
1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL11242 |
| Series (1) |
| GSE25561 |
Role of chaperones and a cytoplasmatic protease in recombinant protein production in E. coli at suboptimal growth temperature |
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