Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.
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.
Uchiyama
Taku
T
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Miyazaki
Kentaro
K
D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D054892:Metagenome; D056186:Metagenomics / Q000379:methods*; D009876:Operon
D049452:Green Fluorescent Proteins; D004247:DNA