Sean Clarke/Paper notes: Difference between revisions
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Revision as of 10:52, 15 January 2007
Light stimulates growth of proteorhodopsin-containing marine Flavobacteria [1] Alphaproteobacteria and Gammaproteobacteria, together with members of the Bacteroidetes phylum, are the most abundant groups of heterotrophic bacteria in the sea.
From supplemental info: Annotation and phylogenetic analysis. Open reading frames were predicted and autoannotated using GenDB (Meyer et al. 2003). In addition, all relevant genes discussed here were manually annotated. For the phylogenetic analysis of PR, amino acid sequences were aligned using Clustal W, and a tree was constructed based on a Kimura’s distance matrix and the Neighbour-Joining method using the PHYLIP package (Version 3.2) (Felsenstein 1989).
For bacterial counts, samples were fixed with 0.2 μm-pore-size filtered formaldehyde (4%, final concentration), stained with SYBR Gold (1:100 dilution, Molecular Probes), filtered onto black 0.2 μm-pore-size polycarbonate filters (Poretics, Osmonics Inc.) and counted by epifluorescence microscopy within 48 hours.
RNA extraction and purification. Cells were harvested from seawater cultures by pipetting 10 ml samples into 15-ml tubes on ice. Samples were centrifuged and the pellets were stored with 0.5 ml RNAlater® (Ambion Inc., Austin, TX) at –80 oC. For RNA extraction, samples were thawed on ice. The RNAlater was discarded and the pellet was washed in PBS 1X. A total of 500 μl of lysis/binding solution provided by the RNAqueous®-4PCR kit (Ambion, Inc.) were added to the cells. The samples were transferred to 2 ml screw-cap microcentrifuge tubes containing 1.2 g of 100-μm-diameter zirconia-silica beads (BioSpec Products, Inc.). Samples were mechanically disrupted in a Mini-beadbeater-8TM cell disrupter (BioSpec Products Inc., Bartlesville, OK). After disruption, samples were incubated on ice for 5 min and the beads were allowed to settle out of the lysis mixture. The lysate was clarified by centrifugation and the aqueous phase was transferred to a new tube. An equal volume of 64% ethanol was added to the lysate and samples were purified according to the RNAqueous®- 4PCR Kit. The isolated total RNA was treated with DNase I - RNase-free (Ambion Inc.). RNA preparations were checked for DNA contamination with PCR using primers 358F and 907RM for the 16S rRNA gene. Total RNA was quantified by spectrophotometry at 260 nm.