title: Directed evolution of λ integrase activity and specificity by genetic derepression.
abstract: Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using β-lactamase and the β-lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin. Using this method, we generated λ integrase variants displaying improved in vitro recombination of a non-cognate substrate present in the human genome. One generalist integrase variant displaying enhanced catalytic activity was further used in a facile, single-step transformation method to introduce transgenes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.
authors: p53 Laboratory, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore.
authors: Jia Wei
authors: JW
authors: Siau
authors: p53 Laboratory, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore.
authors: Sharon
authors: S
authors: Chee
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors: Harshyaa
authors: H
authors: Makhija
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors: Cho Mar Myint
authors: CM
authors: Wai
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors: Shree Harsha Vijaya
authors: SH
authors: Chandra
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors: Sabrina
authors: S
authors: Peter
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors: Peter
authors: P
authors: Dröge
authors: p53 Laboratory, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore faridg@p53Lab.a-star.edu.sg.
authors: Farid J
authors: FJ
authors: Ghadessy
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D019426:Integrases; D001618:beta-Lactamases
keywords: directed evolution; integrase; recombineering; synthetic biology