Computational protocol: Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

Similar protocols

Protocol publication

[…] To evaluate the binding affinity and function of carbohydrate binding modules in the cellobiohydrolase Ra3055, truncational variants were created and tested for both catalytic and binding activity. The nucleotide sequences encoding the truncated variants of Ra3055 were amplified by PCR with the primers listed on . The PCR amplicon was cloned into the pET46 vector using ligation independent cloning as described above. The recombinant proteins from each cell lysate containing the truncated variant was purified by a three step chromatography procedure as described above, and the purity was assessed by SDS/PAGE. The protein concentrations were calculated based on the molecular mass and computed extinction coefficients. The extinction coefficients for TM1, TM2, TM3 and TM4 were 198,270 M−1cm−1, 226,020 M−1cm−1, 172,830 M−1cm−1 and 158,390 M−1cm−1 respectively. The enzymatic activities of each truncational variant on cellotetraose and on cellulose (PASC and Avicel) were determined as described above. [...] The total RNA was extracted from R. albus 8 cells grown in a defined medium containing either cellobiose or phosphoric acid swollen cellulose (PASC). The cells were harvested at mid-log phase by combining the culture with 2 volumes of RNAprotect® bacterial reagent (Qiagen), followed by centrifugation at 13,000 × g for 10 min at room temperature. The cell pellets were stored at −80 °C until RNA extraction. In the subsequent steps, the cell pellets were treated with lysis buffer (200 U/ml of mutanolysin, 150 μg/ml of proteinase K, 25 mM EDTA, and 0.5% SDS) for 30 minute at 55 °C. The total RNA was extracted with the RNeasy mini kit (Qiagen) with the optional on-column DNase treatment step. Then, the total RNA was eluted with DEPC-treated nuclease-free water and bacterial ribosomal RNAs were removed with the MicrobExpress kit (LifeTechnologies). The enriched mRNA fraction was converted to RNA-Seq libraries using the TruSeq Stranded RNA Sample Prep kit from Illumina. The expression of cellobiohydrolases and cellobiose phosphorylases were analyzed using CLC Genomics Workbench version 5.5.1 (CLC Bio-Qiagen, Aarhus, Denmark). The genomic sequence of R. albus 8 (NZ_ADKM020000001 to NZ_ADKM02000136) was used as the reference genome, and the reads were mapped onto the reference sequences using the CLC software. Reads were only assembled if the fraction of the read that aligned with the reference genome was greater than 0.9 and if the read matched other regions of the reference genome at less than 10 nucleotide positions. […]

Pipeline specifications

Software tools TM4, PASC, CLC Genomics Workbench
Applications Phylogenetics, RNA-seq analysis
Organisms Ruminococcus albus, Homo sapiens, Bos taurus
Chemicals Cellobiose, Glucose