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Nat Protoc. 2008;3(7):1202-12. doi: 10.1038/nprot.2008.96.

Substrate-induced gene expression (SIGEX) screening of metagenome libraries.

Author information

1
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8561, Japan. uchiyama-taku@aist.go.jp

Abstract

Substrate-induced gene-expression screening (SIGEX) has been developed for isolating novel catabolic genes from environmental metagenomes, particularly genes that are difficult to obtain using conventional gene-cloning methods. In SIGEX, restriction enzyme-digested metagenome fragments are ligated into an operon-trap vector (e.g., p18GFP), and a library is constructed in a liquid culture by transforming a cloning host (e.g., Escherichia coli). The library is subjected to a substrate-dependent gene-induction assay, and positive cells are selected by detecting activity of a co-expressed marker (e.g., GFP) encoded in the vector. High-throughput screening is possible if fluorescence-activated cell sorting (FACS) is used to select GFP-expressing cells. The abovementioned SIGEX procedure requires approximately 17 d. In this protocol, a widely applicable SIGEX scheme is presented along with typical experimental results.

PMID:
18600226
DOI:
10.1038/nprot.2008.96
[Indexed for MEDLINE]

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