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 http://www.expasy.org/tools/dna.html. Sequence predictions were retrieved from the protein and nucleotide databases of the National Center for Biotechnology Information (NCBI) Entrez page http://www.ncbi.nlm.nih.gov/Entrez/. 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)