Dataset Information

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

ABSTRACT: We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

SUBMITTER: Baba T

PROVIDER: S-EPMC1681482 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Baba Tomoya T Ara Takeshi T Hasegawa Miki M Takai Yuki Y Okumura Yoshiko Y Baba Miki M Datsenko Kirill A KA Tomita Masaru M Wanner Barry L BL Mori Hirotada H

Molecular systems biology 20060221

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-t ...[more]

PMID: 16738554

Similar Datasets

Project description:BackgroundIt is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth.ResultsIn the present study, we considered the simulation of the main metabolic pathways such as glycolysis, TCA cycle, pentose phosphate (PP) pathway, and the anapleorotic pathways using enzymatic reaction models of E. coli. Once intracellular fluxes were computed by this model, the specific ATP production rate, the specific CO? production rate, and the specific NADPH production rate could be estimated. The specific ATP production rate thus computed was used for the estimation of the specific growth rate. The CO? production rate could be used to estimate cell yield, and the specific NADPH production rate could be used to determine the flux of the oxidative PP pathway. The batch and continuous cultivations were simulated where the changing patterns of extracellular and intra-cellular metabolite concentrations were compared with experimental data. Moreover, the effects of the knockout of such pathways as Ppc, Pck and Pyk on the metabolism were simulated. It was shown to be difficult for the cell to grow in Ppc mutant due to low concentration of OAA, while Pck mutant does not necessarily show this phenomenon. The slower growth rate of the Ppc mutant was properly estimated by taking into account the lower specific ATP production rate. In the case of Pyk mutant, the enzyme level regulation was made clear such that Pyk knockout caused PEP concentration to be up-regulated and activated Ppc, which caused the increase in MAL concentration and backed up reduced PYR through Mez, resulting in the phenotypic growth characteristics similar to the wild type.ConclusionsIt was shown to be useful to simulate the main metabolism of E. coli for understanding metabolic changes inside the cell in response to specific pathway gene knockouts, considering the whole main metabolic pathways. The comparison of the simulation result with the experimental data indicates that the present model could simulate the effect of the specific gene knockouts to the changes in the metabolisms to some extent.

Dataset Information

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Publications

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets