Computational protocol: First co-expression of a lipase and its specific foldase obtained by metagenomics

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Protocol publication

[…] The clone with the genes lipg9 and lifg9, for the lipase and foldase, respectively, was previously isolated from a metagenomic DNA library built from a fat-contaminated soil []. The plasmid DNA was isolated, by the alkaline lysis method [], for template usage for PCR and for DNA sequencing. For the latter, a collection of derivative plasmids containing randomly inserted EZ-Tn5 < KAN-2 > was obtained by using an in vitro transposon insertion reaction with the EZ-Tn5 < KAN-2 > Insertion Kit (Epicentre). These derivative plasmids were then used to generate new clones. Genes from 96 inactive clones were then sequenced with the ABI 377 (Genetic Analyzer, Applied Biosystems/HITACHI, Foster City, CA, USA) and MegaBACE 1000 (GE Healthcare, Uppsala, Sweden) sequencers, from both ends, using DYEnamic ET Dye Terminator Kit (GE Life Sciences). Sequence assembly and editing were performed with the CodonCode Aligner software (CodonCode Corporation, Centerville, MA, USA). The open reading frames (ORFs) were identified with the ORF Finder tool (NCBI) []. […]

Pipeline specifications

Software tools CodonCode Aligner, Open Reading Frame Finder
Application Metagenomic sequencing analysis
Organisms Aeromonas veronii, Escherichia coli BL21(DE3)
Chemicals Calcium, Triolein