title: Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA.
abstract: Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.
authors.affiliation: Medical Division, The Wakasa Wan Energy Research Center, Tsuruga 914-0192, Japan.
authors.forename: Go
authors.initials: G
authors.lastname: Kagiya
authors.forename: Ryohei
authors.initials: R
authors.lastname: Ogawa
authors.forename: Masanori
authors.initials: M
authors.lastname: Hatashita
authors.forename: Keiichi
authors.initials: K
authors.lastname: Takagi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kodaki
authors.forename: Shingo
authors.initials: S
authors.lastname: Hiroishi
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics*; D019143:Evolution, Molecular; D024861:Genetic Enhancement / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins