Open AccessConstruction of an insertional-inactivation cloning vector for Escherichia coli using a Rhodococcus gene for indigo production
Author(s) -
Stephen L. Hart,
David R. Woods
Publication year - 1992
Publication title -
journal of general microbiology/journal of general microbiology
Language(s) - English
Resource type - Journals
eISSN - 2059-9323
pISSN - 0022-1287
DOI - 10.1099/00221287-138-1-205
Subject(s) - ecori , plasmid , multiple cloning site , biology , escherichia coli , cloning (programming) , molecular cloning , genetics , insertional mutagenesis , cloning vector , indigo , gene , microbiology and biotechnology , expression vector , recombinant dna , complementary dna , mutant , computer science , programming language , art , visual arts
pSLH8, an insertional-inactivation cloning vector for Escherichia coli has been constructed by inserting a pigment gene (probably encoding an indole dioxygenase) from Rhodococcus sp. ATCC 21145 into pUC18. Wild-type E. coli colonies containing pSLH8 produce insoluble indigo and turn dark blue on unsupplemented LB agar. Insertion of DNA fragments into the unique BamHI, EcoRI, EcoRV, HindIII, PstI, SphI and SstI polylinker cloning sites disrupts the reading frame of the fully sequenced pigment gene and results in unpigmented colonies. pSLH8 may be an attractive alternative to pUC18 and similar plasmids because it does not require specifically mutated host strains or an expensive substrate for colour development.