Versatile broad-host-range cosmids for construction of high quality metagenomic libraries

• Constructed two low-copy broad-host-range Gateway® entry cosmids
• Presented a detailed protocol of constructing high-quality metagenomic library
• Cloned > 33 kb random metagenomic DNA in the cosmids
• Generated metagenomic DNA libraries of up to 9 × 106 clones
• Transferred the cloned HMW DNA from the cosmids to Gateway® destination vectors

We constructed IncP broad-host-range Gateway® entry cosmids pJC8 and pJC24, which replicate in diverse Proteobacteria. We demonstrate the functionality of these vectors by extracting, purifying, and size-selecting metagenomic DNA from agricultural corn and wheat soils, followed by cloning into pJC8. Metagenomic DNA libraries of 8 × 104 (corn soil) and 9 × 106 (wheat soil) clones were generated for functional screening. The DNA cloned in these libraries can be transferred from these recombinant cosmids to Gateway® destination vectors for specialized screening purposes. Those library clones are available from the Canadian MetaMicroBiome Library project (http://www.cm2bl.org/).