An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus.
A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.
Tamakoshi
M
M
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Japan.
Yaoi
T
T
Oshima
T
T
Yamagishi
A
A
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D017353:Gene Deletion; D018390:Gene Targeting; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D020161:Physical Chromosome Mapping; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D009962:Orotate Phosphoribosyltransferase