Ez tn5 kan 2 insertion kit

To clone the recA genomic island (RME) from V. cholerae strain S24, genomic DNA was digested with NaeI and a library constructed using the CopyControl Fosmid Library Production Kit (Epicentre). NaeI digestion of V. cholerae strain S24 genomic DNA creates a fragment of 38, 913 bp containing the entire 32, 787 bp RME. The library was screened for a fosmid clone containing the 38, 913-bp NaeI fragment using primers targeting the phage integrase in the RME (Table 1). A positive clone designated pCC2FOS-RME was confirmed by sequencing the ends of the cloned insert using the pCC2FOS vector primers FP and RP (Table 1). To create the pCC2FOS no insert control, linearized and dephosphorylated pCC2FOS (Epicentre) was treated with T4 polynucleotide kinase and circularized by ligation. A mutant library of pCC2FOS-RME was constructed using the EZ-Tn5 Kan-2 Insertion Kit (Epicentre Biotechnologies) according to manufacturer instructions. Mutants containing knockouts of individual genes present on the genomic island were screened by PCR using primers reading out from EZ-Tn5 Kan-2 and a primer targeting the gene of interest (Table 1).

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 [24 (link)]. The plasmid DNA was isolated, by the alkaline lysis method [83 ], 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) [84 (link)].

Escherichia coli EPI300™-T1R and pCC2FOS fosmid vector (CopyControl™ Fosmid Library Production Kit, Epicentre Biotechnologies, Madison, USA) were used for constructing the metagenomic library [24 (link)]. EZ-Tn5 < KAN-2 > Insertion Kit (Epicentre) and DYEnamic ET Dye Terminator Kit (GE Life Sciences, Uppsala, Sweden) were used for sequencing. The NucleoSpin®Extract II PCR purification kit was from Macherey-Nagel GmbH & Co (Düren, Germany). The strains E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) and BL21(DE3) (Novagen, Madison, MI, USA) and the vectors pET-28a(+), pET-29b(+) (also from Novagen) and pT7-7 (Addgene, Cambridge, MA, USA) were used as the recombinant protein expression system.