Computational protocol: Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches

Similar protocols

Protocol publication

[…] For the molecular identification of investigated Thermobifida strains PCR amplification of 16S rDNAs were performed as described by Rainey et al. []. Partial sequences of the first 500 bp of the 16S rDNA were initiated with the 531r conservative eubacterial primer, almost-complete 16S rDNA sequences were determined by using primers 27f, 531r, 803f and 1492r []. 16S rDNA sequence reads and amino acid sequences of the cloned endomannanases were assembled in MEGA6 [] then aligned by using the ClustalW algorithm. Neighbor-joining trees were constructed in MEGA5, performing 1000 bootstrap replicates. [...] 16SrDNA sequences and DNA fragments up- and downstream of endomannanases subcloned into pUC19 were determined with a DNA sequencer (ABI Prism 310, Perkin Elmer Co., USA).DNA and protein endomannanase sequences were analyzed by using the BLAST server [] and the MEGA6 software package []. Amino acid sequence and domain structure of ManB were determined by Swiss-Prot, EMBL and NCBI database queries and by using the Pfam [] and InterPro [] bioinformatics servers. Phylogenetic trees were reconstructed by the maximum likelihood method [] by using the MEGA6 software package. […]

Pipeline specifications

Software tools MEGA, Clustal W, InterPro
Databases Pfam UniProt
Applications Phylogenetics, Protein sequence analysis
Organisms Thermobifida fusca, Tyto alba