|
| Status |
Public on Feb 18, 2016 |
| Title |
growth rate 0.15 h vs. growth rate 0.3 h, replica 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lactococcus lactis growth rate 0.15 h
|
| Organism |
Lactococcus lactis |
| Characteristics |
subspecies: MG1363
growth rate: 0.15 h
|
| Treatment protocol |
None
|
| Growth protocol |
Glucose-limited chemostat cultures were grown in 2 L bioreactors with a working volume of 1.2 L at 30 °C, under continuous stirring. The headspace was flushed at 5 headspace volume changes per hour, with a gas mixture of 95% N2 (99.998% pure) and 5% CO2 (99.7% pure) with oxygen impurity less than 34 ppm. A pH of 6.5±0.05 was maintained by automatic titration with 5 M NaOH. Fermenters were inoculated with 4% (v/v) of standardized pre-cultures consisting of 45 mL of CDMPC inoculated with 300 µL of a glycerol stock of L. lactis MG 1363 and incubated for 16 h at 30 °C. After batch growth until an optical density at 600 nm (OD600) of around 1.8, medium was pumped at the appropriate dilution rate (0.15, 0.3, 0.5, 0.6 h-1). The actual dilution rate was measured shortly before harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted and washed in resuspension buffer [1 mM Tris-HCl pH 7.2 and 10 mM Magnesium chloride]. Cells were lysed in 2 vol Phenol (saturated with resuspension buffer) by vortexing with glass beads (100 μm diameter) in an ice-cold Tissue lyser (Qiagen). Aqueous Phase was precipitated with 1 vol Isopropanol and 0.1 volume of Potassium acetate (pH 5). Total RNA was pelleted and dissolved in 1 vol 1 M NaCl and reprecipitated with 1 vol Isopropanol. Pellet was resuspended in DNase/RNase-free water.
|
| Label |
Cy3
|
| Label protocol |
1. Equal amounts of in vitro synthesized tRNA standards (16 nM each; E. coli tRNALys2, E. coli tRNATyr2, Yeast tRNAPhe3) were added to 2 µg extracted total RNA prior to deacetylation.
2. Total RNA containing tRNA standards was treated for 45 min at 37°C with 100 mM Tris-HCl (pH 9) for tRNA deacetylation.
3. Cy3 or Atto653 containing oligonucleotides (Microsynth) were ligated to tRNA 3'-CCA ends using 20 U/μl T4 DNA ligase (New England Biolabs) for 16h at 16°C in the presence of 15% DMSO (Sigma-Aldrich).
|
| |
|
| Channel 2 |
| Source name |
Lactococcus lactis growth rate 0.3 h
|
| Organism |
Lactococcus lactis |
| Characteristics |
subspecies: MG1363
growth rate: 0.3 h
|
| Treatment protocol |
None
|
| Growth protocol |
Glucose-limited chemostat cultures were grown in 2 L bioreactors with a working volume of 1.2 L at 30 °C, under continuous stirring. The headspace was flushed at 5 headspace volume changes per hour, with a gas mixture of 95% N2 (99.998% pure) and 5% CO2 (99.7% pure) with oxygen impurity less than 34 ppm. A pH of 6.5±0.05 was maintained by automatic titration with 5 M NaOH. Fermenters were inoculated with 4% (v/v) of standardized pre-cultures consisting of 45 mL of CDMPC inoculated with 300 µL of a glycerol stock of L. lactis MG 1363 and incubated for 16 h at 30 °C. After batch growth until an optical density at 600 nm (OD600) of around 1.8, medium was pumped at the appropriate dilution rate (0.15, 0.3, 0.5, 0.6 h-1). The actual dilution rate was measured shortly before harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted and washed in resuspension buffer [1 mM Tris-HCl pH 7.2 and 10 mM Magnesium chloride]. Cells were lysed in 2 vol Phenol (saturated with resuspension buffer) by vortexing with glass beads (100 μm diameter) in an ice-cold Tissue lyser (Qiagen). Aqueous Phase was precipitated with 1 vol Isopropanol and 0.1 volume of Potassium acetate (pH 5). Total RNA was pelleted and dissolved in 1 vol 1 M NaCl and reprecipitated with 1 vol Isopropanol. Pellet was resuspended in DNase/RNase-free water.
|
| Label |
Atto635
|
| Label protocol |
1. Equal amounts of in vitro synthesized tRNA standards (16 nM each; E. coli tRNALys2, E. coli tRNATyr2, Yeast tRNAPhe3) were added to 2 µg extracted total RNA prior to deacetylation.
2. Total RNA containing tRNA standards was treated for 45 min at 37°C with 100 mM Tris-HCl (pH 9) for tRNA deacetylation.
3. Cy3 or Atto653 containing oligonucleotides (Microsynth) were ligated to tRNA 3'-CCA ends using 20 U/μl T4 DNA ligase (New England Biolabs) for 16h at 16°C in the presence of 15% DMSO (Sigma-Aldrich).
|
| |
|
| |
| Hybridization protocol |
1. Fluorescently labeled tRNAs were hybridized on the microarrays for 16 h at 60°C in a Hyb4 microarray hybridization system (Digilab) supplemented with polyA (0.17 mg/ml) and salmon sperm DNA (0.17 mg/ml).
2. Microarrays were washed once in 2× SSC/0.1% SDS (50°C), once in 1× SSC/0.1% SDS (42°C) and then three times in 0.1× SSC (42°C).
3. Arrays were dried and storred in the dark at 4°C.
|
| Scan protocol |
Arrays were scanned with a GenePIX 4200A (Molecular Devices) instrument.
|
| Description |
0.15_0.3_rep1
|
| Data processing |
1. TIF-files of scanned arrays were analyzed using the GenePix Pro 7 (Molecular Devices) software.
2. Ratios of Cy3/Atto635 fluorescence signals were calculated for individual spots based on median fluorescence signal (background fluorescence subtraction).
3. Cy3/Atto635 ratios were normalized to Cy3/Atto635 values of tRNA standards (e.g. median Cy3/Atto635 ratios of the three tRNA standards E. coli tRNALys2, E. coli tRNATyr2, Yeast tRNAPhe3) for individual blocks.
4. Normalized Cy3/Atto635 ratios for individual blocks were averaged, representing final Cy3/Atto635 ratios.
|
| |
|
| Submission date |
Feb 25, 2014 |
| Last update date |
Feb 18, 2016 |
| Contact name |
Paul Georg Saffert |
| E-mail(s) |
psaffert@gmx.de
|
| Organization name |
University of Potsdam
|
| Department |
Biochemistry
|
| Lab |
Ignatova
|
| Street address |
Karl-Liebknecht Straße 24-25
|
| City |
Potsdam |
| State/province |
Brandenburg |
| ZIP/Postal code |
14476 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18332 |
| Series (1) |
| GSE55336 |
Relative tRNA abundance in Lactococcus lactis as a function of growth rate and overexpression stress |
|
