title: Rapid one-step inactivation of single or multiple genes in Escherichia coli.
abstract: Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D-1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.
authors: Department of Chemical and Biomolecular Engineerin-BK21 Program, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology-KAIST, Daejeon, Republic of Korea.
authors: Chan Woo
authors: CW
authors: Song
authors: Sang Yup
authors: SY
authors: Lee
mesh_terms: D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D005798:Genes, Bacterial; D005819:Genetic Markers / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D045522:Recombinases / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D029968:Escherichia coli Proteins; D005650:Fumarates; D005819:Genetic Markers; D045522:Recombinases; C032005:fumaric acid
keywords: Cre recombinase; Escherichia coli; Gene manipulation; Metabolic engineering; Red recombinase