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 ∼17 d. In this protocol, a widely applicable SIGEX scheme is presented along with typical experimental results.
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Acknowledgements
This work was supported by the Japan Society for Promotion of Science. We thank Robert A. Kanaly for critical reading of this manuscript.
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T.U. prepared the experimental data and wrote the paper, and K.W. wrote the paper.
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Uchiyama, T., Watanabe, K. Substrate-induced gene expression (SIGEX) screening of metagenome libraries. Nat Protoc 3, 1202–1212 (2008). https://doi.org/10.1038/nprot.2008.96
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DOI: https://doi.org/10.1038/nprot.2008.96
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