RADOM, an Efficient In Vivo Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces cerevisiae

被引：34

作者：

Lin, Qiuhui ^{[1
,2
]}

Jia, Bin ^{[1
,2
]}

Mitchell, Leslie A. ^{[3
,4
]}

Luo, Jingchuan ^{[3
,4
]}

Yang, Kun ^{[3
,5
,6
]}

Zeller, Karen I. ^{[3
]}

Zhang, Wenqian ^{[1
,2
]}

Xu, Zhuwei ^{[3
,4
]}

Stracquadanio, Giovanni ^{[3
,5
,6
]}

Bader, Joel S. ^{[3
,5
,6
]}

Boeke, Jef D. ^{[3
,4
]}

Yuan, Ying-Jin ^{[1
,2
]}

机构：

[1] Tianjin Univ, Key Lab Syst Bioengn, Minist Educ, Tianjin 300072, Peoples R China

[2] Sch Chem Engn & Technol, Collaborat Innovat Ctr Chem Sci & Engn Tianjin, Tianjin 300072, Peoples R China

[3] Johns Hopkins Univ, Sch Med, Inst Basic Biomed Sci, High Throughput Biol Ctr, Baltimore, MD 21205 USA

[4] NYU, Langone Med Ctr, Inst Syst Genet, New York, NY 10016 USA

[5] Johns Hopkins Univ, Dept Biomed Engn, Baltimore, MD 21218 USA

[6] Johns Hopkins Univ, Inst Computat Biol, Baltimore, MD 21218 USA

来源：

ACS SYNTHETIC BIOLOGY | 2015年 / 4卷 / 03期

基金：

加拿大自然科学与工程研究理事会; 美国国家科学基金会;

关键词：

in vivo assembly; synthetic yeast genome; Sc2.0; Build-A-Genome; synthetic biology; HOMOLOGOUS RECOMBINATION; YEAST-CELLS; GENE; CONSTRUCTION; STRATEGY; VECTORS; CLONING; VITRO;

D O I：

10.1021/sb500241e

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeas to with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble similar to 3 and similar to 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 similar to 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.

引用

页码：213 / 220

页数：8