Versatile broad-host-range cosmids for construction of high quality metagenomic libraries.
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 8×10(4) (corn soil) and 9×10(6) (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/).
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Jiujun
J
Cheng
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Lee
L
Pinnell
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Katja
K
Engel
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Josh D
JD
Neufeld
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada. Electronic address: trevor.charles@uwaterloo.ca.
Trevor C
TC
Charles
D003360:Cosmids; D015723:Gene Library; D005827:Genetics, Microbial / Q000379:methods*; D058507:Host Specificity; D056186:Metagenomics / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D020560:Proteobacteria / Q000235:genetics*
Broad-host-range cosmids; DNA extraction; Functional metagenomics; Gateway® recombination vector; Metagenomic libraries; Soil