Computational protocol: The regulatory function of LexA is temperature-dependent in the deep-sea bacterium Shewanella piezotolerans WP3

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Protocol publication

[…] The cells used to assess the cold shock response were prepared as described previously (). The samples were harvested by centrifugation and immediately placed in liquid nitrogen prior to RNA extraction. The WP3 strains were inoculated into 2216E medium at different temperatures as indicated in the text, and the culture was collected immediately when the cells reached the mid-exponential phase (OD600≈0.8). Total RNA extraction, reverse transcription and real-time quantitative real-time PCR (qPCR) were performed as described previously (). The primer pairs (Table ) used to amplify the selected genes in qPCR were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). [...] A microarray that contained 95% of the total predicted gene content of WP3 was designed and manufactured (CapitalBio, Beijing, China). The preparation of fluorescent dye-labeled DNA and hybridization image acquisition, data processing and clustering were performed as previously described (). Briefly, the total RNA was reverse transcribed with SuperScript II (Invitrogen, Carlsbad, CA, USA), and the cDNAs were labeled with Cy3 and Cy5 using the Klenow enzyme (Takara Bio Inc., Japan) according to the manufacturers’ instructions. Labeled cDNA was purified with the PCR purification kit (Macherey-Nagel, Düren, Germany) and resuspended in elution buffer. The microarray slides were hybridized with cDNA prepared from 3 biological replicate samples. As a measure of technical replication, the dye-swap experiment was performed on each sample so that a total of six data points were available for every ORF on the microarray. A LuxScan 10K scanner and microarray scanner 2.3 software (CapitalBio, Beijing, China) were used for the array image acquisition. The linear normalization method was used for data analysis based on the expression levels of WP3 housekeeping genes in combination with the yeast external controls. The normalized data were log-transformed and loaded into MAANOVA under the R environment for multiple testing by fitting a mixed-effects ANOVA model (). Microarray spots with P values <0.001 in the F-test were regarded as DEGs. All of the DEGs were confirmed with the Significance Analysis of Microarrays (SAM) software (). [...] The SOS Box was identified by searching the 5′ regions (1–700 bp upstream of the start codon) of the genes in WP3 using the conservative sequence of SOS box in γ-proteobacterium. The information matrix for the generation of the LexA Logo was produced by aligning the WP3 LexA binding sequences predicted by the RegPredict web server, which is available at http://regpredict.lbl.gov (). A graphical representation of the matrix through a Logo graph was obtained with Weblogo software, which is available at http://weblogo.berkeley.edu (). […]

Pipeline specifications

Software tools R/maanova, SAM, RegPredict, WebLogo
Applications Gene expression microarray analysis, Genome data visualization
Organisms Escherichia coli, Shewanella piezotolerans WP3, Shewanella piezotolerans
Chemicals Dimethyl Sulfoxide