|
| Status |
Public on Aug 06, 2013 |
| Title |
PCC6803_producer_4d_copper_depletion 309_4d-2 |
| Sample type |
RNA |
| |
|
| Source name |
PCC6803 producer 4d copper depletion
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
strain: ethanol producer
time: 4d
treatment: copper depletion
|
| Treatment protocol |
Induction of ethanol synthesis from the petJ promoter in #309 was triggered by centrifugation and resuspension of pre-cultures in copper-free medium. Thereby, pre-cultures of OD750 = 7-8 were diluted to a final OD750 of 2 (equivalent to about 10mg chlorophyll * L-1) and subsequently divided into triplicates. In order to maintain maximal ethanol production, nutrient limitations were countersteered by proportionate supplementation of a 100x nutrient concentrate when the nitrate concentration was below 50% of the BG11 concentration.
|
| Growth protocol |
The ethanologenic strain #309 of Synechocystis 6803 and the isogenic wild-type control strain #621 were cultivated in triplicate for 19 days in optimized photobioreactors (PBRs) containing 0.5 L BG11 medium (Rippka et al., 1979) supplemented with 2 mM TES, 35 g/L “instant ocean” seawater salts (Aquarium Systems Inc., France) and 10 µg/ml gentamycin. The lid was fitted with ports for incoming pH-, dissolved oxygen- and temperature-sensors as well as sampling ports and connections to in- and out-gas. Dissolved oxygen, pH and temperature were monitored by three-channel MultiMeter 44 devices (Crison Instruments, S.A. Spain). Cells were grown under day-night cycle conditions with a 12h photoperiod. The light intensity was successively adapted to the increasing cell density (~100 µE m-2 s-1 per OD750 unit) and reached a maximum value of 1000 µE m-2 s-1. The culture temperature was controlled in a day-night cycle with 35°C day-time and 25°C night-time temperature. During the 12h photoperiod, the liquid phase was discontinuously aerated with CO2 enriched air (10% CO2) pH-dependent and computer-controlled: At a culture pH above 7.35, the aeration started and incoming air flow ceased at a pH below 7.25. There was no aeration of the culture at night. Cells were constantly (24h) mixed by stirring with a magnetic stir bar (7 cm length) at 250 rpm. Samples from discrete stages of cultivation were subsequently subjected to microarray (transcriptome) analysis. Furthermore, growth, ethanol accumulation, pigment profiles and photosynthetic capacity (not shown) were monitored over the cultivation period.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples from discrete stages of cultivation in PBRs were immediately quenched on ice and spun down at 0 °C. RNA isolationwas performed essentially as described previously (Dienst et al., 2008)
|
| Label |
Cy3
|
| Label protocol |
The RNA was labeled directly, without cDNA synthesis in 2 µg aliquots with the Kreatech “ULS labeling kit for Agilent gene expression arrays” with Cy3 according to the manufacturers protocol.
|
| |
|
| Hybridization protocol |
The labelled RNA was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays with 1.65 µg of labeled RNA
|
| Scan protocol |
Arrays were scanned on the Agilent Technologies Scanner G2505B, using Agilent Feature Extraction Software 10.7.3.1 and the protocol GE1_107_Sep09 for Cy3 labelled arrays
|
| Data processing |
Raw data were processed with the R package Limma. Median signal intensity was quantile normalized.
|
| |
|
| Submission date |
Aug 05, 2013 |
| Last update date |
Aug 06, 2013 |
| Contact name |
Jens Georg |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Freiburg
|
| Street address |
Schänzlestr. 1
|
| City |
Freiburg |
| ZIP/Postal code |
79104 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15867 |
| Series (1) |
| GSE49552 |
Transcriptomic response to long time ethanol production in the cyanobacterium Synechocystis sp. PCC6803 |
|
