Abstract
The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an “operon-trap” vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59, 143–169.
Cowan, D. A. (2000) Microbial genomes – the untapped resource. Trends Biotechnol 18, 14–16.
Handelsman, J., Rondon, M. R., Brady, S. F., Clardy, J., and Goodman, R. M. (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5, 245–249.
Lorenz, P., Liebeton, K., Niehaus, F., and Eck, J. (2002) Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. Curr Opin Biotechnol 13, 572–577.
Ferrer, M., Martínez-Abarca, F., and Golyshin, P. N. (2005) Mining genomes and ‘metagenomes’ for novel catalysts. Curr Opin Biotechnol 16, 1–6.
Uchiyama, T., Abe, T., Ikemura, T., and Watanabe, K. (2005) Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23, 88–93.
Uchiyama, T. and Watanabe, K. (2007) The SIGEX scheme: high throughput screening of environmental metagenomes for the isolation of novel catabolic genes. Biotechnol Genet Eng Rev 24, 107–116.
Uchiyama, T. and Watanabe, K. (2008) Substrate-induced gene expression (SIGEX) screening of metagenome libraries. Nat Protoc 3, 1202–1212.
Uchiyama, T. and Watanabe, K. (2006) Improved inverse PCR scheme for metagenome walking. BioTechniques 41, 183–188.
Sambrook, J. and Russell, D. W. (2001) Transformation of E. coli by electroporation. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 1.119–1.122.
Sambrook, J. and Russell, D. W. (2001) Agarose gel electrophoresis. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 5.4–5.13.
Sambrook, J. and Russell, D. W. (2001) Detection of DNA in agarose gels. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 5.14–5.17.
Sambrook, J. and Russell, D. W. (2001) Commonly used techniques in molecular cloning. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. A8.12–A8.16.
Sambrook, J. and Russell, D. W. (2001) Preparation of plasmid DNA by alkaline lysis with SDS. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 1.31–1.42.
Sambrook, J. and Russell, D. W. (2001) DNA sequencing. In: Sambrook, J. and Russell, D. W. Ed. Molecular Cloning: A Laboratory Manual, 3rd Edn., Vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 12.1–12.120.
Rothmel, R. K., Aldrich, T. L., Houghton, J. E., Coco, W. M., Ornston, L. N., and Charkrabaty, A. M. (1990) Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family. J Bacteriol 172, 922–931.
Cowles, C. E., Nichols, N. N., and Harwood, C. S. (2000) BenR, a XylS homolog, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J Bacteriol 182, 6339–6346.
Acknowledgements
This work was supported by the Japan Society for Promotion of Science (No. 19880040).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Uchiyama, T., Miyazaki, K. (2010). Substrate-Induced Gene Expression Screening: A Method for High-Throughput Screening of Metagenome Libraries. In: Streit, W., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 668. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-823-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-60761-823-2_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-822-5
Online ISBN: 978-1-60761-823-2
eBook Packages: Springer Protocols