Computational protocol: Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea

Similar protocols

Protocol publication

[…] The DNA extract was separated by pulsed-filed gel electrophoresis (PFGE), and DNA bands of ∼35 kbp in the gel were extracted by gelase enzymolysis and ethanol precipitation. A metagenomic DNA library was constructed using the CopyControl Fosmid Library Production Kit (Epicentre Biotechnologies, Madison, WI, USA) by following the manufacturer’s instructions. A total of 7,200 fosmid clones were obtained, which were spread onto Luria-Bertani (LB) agar plates supplemented with 1% (v/v) emulsified tributyrin to screen clones with lipolytic enzyme activity. Clones exhibiting a clear zone around the colony were selected to construct their respective subcloning libraries. Fosmid DNA was extracted from positive clones and partially digested by the restriction enzyme Sau3AI. The DNA fragments of 1.5–5 kbp were recovered from an agarose gel, end-repaired and ligated into the pUC19 vector that had been digested by BamHI and pretreated by bacterial alkaline phosphatase. The ligated products were transformed into E. coli TOP10 cells, and the transformants were spread onto LB agar plates supplemented with 100 μg/ml ampicillin and 1% (v/v) tributyrin. Transformants forming clear zones were sequenced. The open reading frames (ORFs) in the sequenced DNA fragments were predicted by the GeneMark program and the genes encoding potential lipolytic enzymes were identified by using the blastx program against the NCBI non-redundant protein database (nr). Multiple sequence alignment was performed using MUSCLE (). Phylogenetic analysis was carried out with the neighbor-joining method using MEGA 6.0 (). The potential signal peptide sequence was predicted by SignalP 4.0 (). […]

Pipeline specifications

Software tools GeneMark, BLASTX, MUSCLE, MEGA, SignalP
Applications Genome annotation, Metagenomic sequencing analysis, Nucleotide sequence alignment
Chemicals Carbon, Sodium Chloride