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

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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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Figure 1: Schematic representation of the substrate-induced gene expression screening (SIGEX) scheme with an example of cloning of a benzoate-degradative operon fragment from the metagenome from groundwater microbial flora19.
Figure 2: Schematic drawing of the substrate-induced gene expression screening (SIGEX) vector (p18GFP) and positive clone which was obtained by a benzoate-induction scheme (pbzo26).
Figure 3: GFP-expression analysis.
Figure 4: Gating in the flow cytometer analysis and sorting of bacterial populations.
Figure 5: Selection of benzoate-positive clones by cell sorting.

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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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Authors and Affiliations

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

    Taku Uchiyama

  2. Laboratory of Applied Microbiology, Marine Biotechnology Institute, 3-75-1, Heita, Kamaishi, Iwate, Japan

    Kazuya Watanabe

  3. Hashimoto Light Energy Conversion Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

    Kazuya Watanabe

  4. Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo, 153-8904, Japan

    Kazuya Watanabe

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  1. Taku Uchiyama
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  2. Kazuya Watanabe
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T.U. prepared the experimental data and wrote the paper, and K.W. wrote the paper.

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Correspondence to Taku Uchiyama.

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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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