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Bacterial Acid Phosphatase Gene Fusions Useful as Targets for Cloning‐Dependent Insertional Inactivation
Author(s) -
Thaller Maria Cristina,
Berlutti Francesca,
Schippa Serena,
Selan Laura,
Rossolini Gian Maria
Publication year - 1998
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp980009t
Subject(s) - cloning (programming) , biology , multiple cloning site , escherichia coli , gene , lac operon , complementation , plasmid , microbiology and biotechnology , protein fragment complementation assay , genetics , fusion gene , molecular cloning , cloning vector , recombinant dna , expression vector , peptide sequence , mutant , computer science , programming language
The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino‐terminal moieties of the Escherichia coli lacZ gene carrying a multiple‐cloning site flanked by phage‐specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC‐like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning‐dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ‐phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ α‐complementation‐based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ‐ phoC fusion‐based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.