Computational protocol: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

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Protocol publication

[…] Sequence analysis was performed with the BigDye Terminator Cycle sequencing kit on an ABI Prism 3700 DNA analyzer (Applied Biosystems, USA). Protein translation was carried out with the Web-based translation tool at the Expasy homepage Sequence predictions were retrieved from the protein and nucleotide databases of the National Center for Biotechnology Information (NCBI) Entrez page Sequence similarity searches were performed with BLAST 2.0. Amino acid sequence alignment of Undec1A with homologous proteins was conducted with Align X, a component of the Vector NTI suite (Informax, North Bethesda, MD, USA), using the blosum62mt2 scoring matrix. Based on comparison of the deduced amino acid sequence, a gene (fumF) encoding a putative novel fumarase was identified and further characterized. A phylogenetic tree was constructed using the neighbor-joining method with Molecular Evolutionary Genetics Analysis 3.1 (MEGA, Version 4.0) []. A boot-strapping value was used to estimate the reliability of phylogenetic reconstructions (1000 replicates). […]

Pipeline specifications

Software tools Vector NTI Advance, MEGA
Databases ExPASy
Applications Phylogenetics, Protein sequence analysis
Organisms Parabacteroides distasonis ATCC 8503, Escherichia coli BL21(DE3)