Wide host-range cloning for functional metagenomics.
We describe how wide host-range cloning vectors can lead to more flexible and effective procedures to isolate novel genes by screening metagenomic libraries in a range of bacterial hosts, not just the conventionally used Escherichia coli. We give examples of various wide host-range plasmid, cosmid, and BAC cloning vectors and the types of genes and activities that have been successfully obtained to date. We present a detailed protocol that involves the construction and screening of a metagenomic library comprising fragments of bacterial DNA, obtained from a wastewater treatment plant and cloned in a wide host-range cosmid. We also consider future prospects and how techniques and tools can be improved.
Wexler
Margaret
M
School of Biological Sciences, University of East Anglia, Norwich, Norfolk, UK.
Johnston
Andrew W B
AW
D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D054892:Metagenome; D056186:Metagenomics / Q000379:methods*; D012722:Sewage / Q000382:microbiology
D004269:DNA, Bacterial; D012722:Sewage