Open AccessA new Thermus-Escherichia coli shuttle integration vector system
Author(s) -
Masatada Tamakoshi,
Masaya Uchida,
Kazuhiro Tanabe,
Shiro Fukuyama,
Akihiko Yamagishi,
Tairo Oshima
Publication year - 1997
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.179.15.4811-4814.1997
Subject(s) - thermus thermophilus , shuttle vector , biology , plasmid , escherichia coli , thermus , genetics , operon , gene , microbiology and biotechnology , subcloning , restriction map , vector (molecular biology) , thermophile , recombinant dna , bacteria
We established a Thermus thermophilus strain in which the pyrE gene (coding for orotate phosphoribosyltransferase of the pyrimidine biosynthetic pathway) was totally deleted. We also constructed an integration vector, which consisted of the Escherichia coli plasmid vector pBluescript and a 2.1-kb segment of the T. thermophilus leu operon sequence, for the integration of a foreign gene into a chromosome of the thermophile. pyrE and leuB genes were used as probes to test the integration vector. The integration vector pINV, bearing the pyrE gene, transformed the delta pyrE strain at a frequency of 6 x 10(-5) through a single crossover event. The leuB gene could also be used as another marker of the integration vector system. The vector could be integrated at the expected site. By digesting the chromosomal DNA of the T. thermophilus transformants with a unique restriction enzyme, the vector could be recovered into E. coli after the recircularization in vitro. The kanamycin nucleotidyltransferase gene could be successfully expressed in the thermophile by using pINV.