Genefish: a window into targeted bacterial diversity
Résumé
The interest for cultivation -independent molecular approaches that extract DNA directly from the environment is related to their capacity to overcome biases in isolation and in vitro cultivation of bacteria, thus providing new possibilities in the search for knowledge within the "black box" of soil microbiology. However, "classical" metagenomic methods are time-and money-intensive requiring construction and screening of million-clone DNA libraries to cover the huge bacterial diversity. Our objective is to develop a complementary method to the traditional metagenomic approaches, based on the use of a specifically engineered recipient E.coli strain to capture genes from soil bacteria after transformation with metagenome DNA. The so-called "Genefish" method is based on the use of specific sequences cloned into the recipient strain to serve as template for homeologous recombination with corresponding DNA present in the metagenome. In addition, the double cross over event involving the targeted genes leads to the replacement of two inducible lethal genes that kill non recombinant bacteria. Details of the E.coli construction including molecular tricks to precisely regulate expression of the lethal genes and of the lambda phage recombinase gene to increase recombination efficiency will be described as well as preliminary applications for the capture of the widespread nitrate reduction genes among soil bacteria. This "Genefish" technology, characterized by the positive screening of recombinant clones is used in the frame of the "Metaexplore" European project, involving 15 institutional and industrial expert groups from 11 countries as one of the technological and conceptual approach developed for discovering new enzymes with important industrial applications, such as chitinases, ligninases and dehalogenases.
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