title: RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.
abstract: We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.
authors.affiliation: †Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.|‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin, 300072, PR China.
authors.forename: Qiuhui
authors.initials: Q
authors.lastname: Lin
authors.affiliation: †Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.|‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin, 300072, PR China.
authors.forename: Bin
authors.initials: B
authors.lastname: Jia
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|∥Institute for Systems Genetics, New York University Langone Medical Center, 550 First Avenue, New York, New York 10016, United States.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Mitchell
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|∥Institute for Systems Genetics, New York University Langone Medical Center, 550 First Avenue, New York, New York 10016, United States.
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Luo
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|#Department of Biomedical Engineering and Institute for Computational Biology, Johns Hopkins University, Baltimore, Maryland 21218, United States.
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.
authors.forename: Karen I
authors.initials: KI
authors.lastname: Zeller
authors.affiliation: †Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.|‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin, 300072, PR China.
authors.forename: Wenqian
authors.initials: W
authors.lastname: Zhang
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|∥Institute for Systems Genetics, New York University Langone Medical Center, 550 First Avenue, New York, New York 10016, United States.
authors.forename: Zhuwei
authors.initials: Z
authors.lastname: Xu
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|#Department of Biomedical Engineering and Institute for Computational Biology, Johns Hopkins University, Baltimore, Maryland 21218, United States.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Stracquadanio
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|#Department of Biomedical Engineering and Institute for Computational Biology, Johns Hopkins University, Baltimore, Maryland 21218, United States.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bader
authors.affiliation: §High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.|∥Institute for Systems Genetics, New York University Langone Medical Center, 550 First Avenue, New York, New York 10016, United States.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
authors.affiliation: †Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.|‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin, 300072, PR China.
authors.forename: Ying-Jin
authors.initials: YJ
authors.lastname: Yuan
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005822:Genetic Vectors; D016681:Genome, Fungal / Q000235:genetics*; D008957:Models, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D004247:DNA
keywords: Build-A-Genome; Sc2.0; in vivo assembly; synthetic biology; synthetic yeast genome