Substrate-induced gene expression (SIGEX) screening of metagenome libraries.
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.
Uchiyama
Taku
T
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
Watanabe
Kazuya
K
D019295:Computational Biology / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D004262:DNA Restriction Enzymes