title: [Tuning of expression level of the genes of interest located in the bacterial chromosome].
abstract: The new method of construction of the set of E. coli clones, differing in the promoter strength upstream the gene of interest, has been developed and tested using native E. coli MG 1655 lacZ gene as the reporter. This method includes the construction of the promoter-carrying DNA fragment obtained by PCR with consensus P(tac) as a template and the primers that lead to randomization of 4 central nucleotides in the promoter "-35"-region, linking the obtained fragments with the selective marker (Cm(R)) followed by Red-driven integration of the resulted DNA fragments directly in E. coli MG1655 chromosome instead the native lacI-gene and promoter/operator region of lac-operon. Due to direct determination of LacZ-activity in the independently obtained clones-integrants, we have found 14 new promoters (from 44 = 256 possible variants) that differ in their strength up to 100 fold (LacZ-activity in the corresponding strains smoothly varies from 10(2) for the weakest tested promoter up to 10(4) Miller U detected for the initial P(tac)). Sequencing of obtained promoters revealed that randomization of three positions in the "-35"-region is sufficient to obtain representative promoter library that would decrease the total number of potential promoter variants from 256 up to 64. It seems probable that exploiting of the developed method leading to one-step construction the library of clones with varied expression of gene/operon of interest could be useful tool in the modem metabolic engineering for optimization of genes expression.
authors.forename: Zh I
authors.initials: ZhI
authors.lastname: Katashkina
authors.forename: A Iu
authors.initials: AIu
authors.lastname: Skorokhodova
authors.forename: D V
authors.initials: DV
authors.lastname: Zimenkov
authors.forename: A Iu
authors.initials: AIu
authors.lastname: Gulevich
authors.forename: N I
authors.initials: NI
authors.lastname: Minaeva
authors.forename: V G
authors.initials: VG
authors.lastname: Doroshenko
authors.forename: I V
authors.initials: IV
authors.lastname: Biriukova
authors.forename: S V
authors.initials: SV
authors.lastname: Mashko
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D016385:TATA Box / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D001616:beta-Galactosidase