Computational protocol: Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum

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Protocol publication

[…] Total RNA was extracted from the 824WT and 824ccpA strain, which were grown in the pH-controlled P2 medium containing 40 g/L d-glucose and 20 g/L d-xylose as the carbon sources, at four time points: middle exponential (time point M), late exponential (time point L), transition (time point T) and stationary phases (time point S) (Figure  A). For investigating the glucose repression in the 824WT, samples were collected at time point S (d-glucose remained then) and S2 and S3 (d-glucose was exhausted at these two time points) (Additional file A). After centrifugation at 4°C, 5000 × g for 10 min, the cell pellets were collected and frozen immediately using liquid nitrogen and then ground into powder. Total RNA was extracted using Trizol™ (Invitrogen, Carlsbad, CA) and purified using RNeasy™ cleanup kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.For microarray analysis, Algilent custom 60-mer oligonucleotide microarrays were used, in which a 60-mer oligonucleotide probe was used for each gene. Single-color microarray assays were performed by Shanghai Biochip Co. Ltd. (Shanghai, China) according to standard protocols provided by Agilent Technologies. In brief, RNA was reversely transcribed to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA was transcribed with T7 RNA polymerase (New England BioLabs, Beverly, MA), resulting in aminoacyl-UTP (aaUTP, Ambion, Austin, TX)-labeled cRNA. cRNA was further labeled with Cy3 NHS ester (GE healthcare, Waukesha, WI) for both the 824WT and 824ccpA, and then hybridized to the probes (size: 60mer; three technical replicates for each ORF) on 8 × 15K custom 60-mer oligonucleotide chip as described previously []. Arrays were scanned with 5-μm resolution using an Agilent DNA Microarray scanner (G2565BA, Agilent Technologies, CA). The raw data was normalized across all arrays by quantile normalization [] function from Feature Extraction software (Agilent Technologies, Santa Clara, CA) and then analyzed in GeneSpring GX (Agilent Technologies, Santa Clara, CA). To investigate expression fold changes of genes in the 824ccpA versus the 824WT, change-fold of every gene was obtained by dividing the normalized signal intensities of the same gene at each time point. 2-fold change was chosen as a threshold for differential expression. Average linkage hierarchical clustering was performed using Cluster 3.0 [], and the resulting gene clusters were visualized in Java TreeView [,]. […]

Pipeline specifications

Software tools GeneSpring GX, TreeView
Application Gene expression microarray analysis
Organisms Clostridium acetobutylicum
Chemicals Arabinose, Carbon, Glucose, Xylose