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Rapid one‐step inactivation of single or multiple genes in Escherichia coli
Author(s) -
Song Chan Woo,
Lee Sang Yup
Publication year - 2013
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201300153
Subject(s) - recombinase , escherichia coli , plasmid , gene , computational biology , mutant , gene knockout , homologous recombination , biology , transformation (genetics) , cre recombinase , genetics , recombination , transgene , genetically modified mouse
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.