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ACS Synth Biol. 2015 Mar 20;4(3):213-20. doi: 10.1021/sb500241e. Epub 2014 Jun 4.

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

Author information

1
†Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.
2
‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin, 300072, PR China.
3
§High Throughput Biology Center, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States.
4
∥Institute for Systems Genetics, New York University Langone Medical Center, 550 First Avenue, New York, New York 10016, United States.
5
#Department of Biomedical Engineering and Institute for Computational Biology, Johns Hopkins University, Baltimore, Maryland 21218, United States.

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.

KEYWORDS:

Build-A-Genome; Sc2.0; in vivo assembly; synthetic biology; synthetic yeast genome

PMID:
24895839
DOI:
10.1021/sb500241e
[Indexed for MEDLINE]

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