title: RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.
abstract: Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week.
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Wang
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Li
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Ruonan
authors.initials: R
authors.lastname: Jia
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Jia
authors.initials: J
authors.lastname: Yin
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Bian
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Aiying
authors.initials: A
authors.lastname: Li
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.|Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI) and Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.affiliation: Genomics, Biotechnology Center, Technische Universität Dresden, Dresden, Germany.
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.|Genomics, Biotechnology Center, Technische Universität Dresden, Dresden, Germany.
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics*; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D007218:Industrial Microbiology / Q000379:methods; D005810:Multigene Family; D009876:Operon; D010957:Plasmids / Q000235:genetics; D039002:Prophages / Q000235:genetics*; D011995:Recombination, Genetic; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D014764:Viral Proteins