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Expression cloning of different bacterial phosphataseencoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green
Author(s) -
Riccio M.L.,
Rossolini G.M.,
Lombardi G.,
Chiesurin A.,
Satta G.
Publication year - 1997
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.1997.tb03570.x
Subject(s) - cloning (programming) , phenolphthalein , gene , microbiology and biotechnology , gene expression , molecular cloning , biology , biochemistry , chemistry , computer science , programming language
A system for expression cloning of bacterial phosphatase‐encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase‐producing clones using an indicator medium (named TPMG) made of Tryptose‐Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase‐encoding genes (respectively encoding a class A non‐specific acid phosphatase, an acid‐hexose phosphatase and a non‐specific alkaline phosphatase) were shotgun‐cloned from the above libraries, indicating that the TPMG‐based expression cloning system can be useful for rapid isolation of different bacterial phosphataseencoding genes.