Development of a versatile shuttle vector for gene expression in Geobacillus spp.
An improved, versatile shuttle vector has been created for the metabolic engineering of Geobacillus spp. As kanamycin is the most thermo-tolerant of commonly used antibiotics, the gene encoding a thermostable kanamycin nucleotidyltransferase, together with the origin of replication from the G. stearothermophilus plasmid pBST1 were cloned into the Escherichia coli cloning vector pUC18. The resulting vector, named pUCG18, replicated in both organisms and could be transformed with an efficiency of 1 x 10(4) transformants per microg of DNA in G. thermoglucosidasius and was stable up to 68 degrees C with antibiotic selection. It was used to demonstrate expression of the pyruvate decarboxylase (pdc) gene from Zymomonas palmae in G. thermoglucosidasius at 45 degrees C. Sequence analysis of the pBST1 derived origin of replication revealed homology with a family of theta replicons that have previously only been found in strains of Bacillus megaterium.
Taylor
Mark P
MP
Division of Biology, Department of Life Sciences, Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK.
Esteban
Carlos D
CD
Leak
David J
DJ
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase