Parallel on-chip gene synthesis and application to optimization of protein expression

被引:113
作者
Quan, Jiayuan [1 ,2 ]
Saaem, Ishtiaq [1 ,2 ]
Tang, Nicholas [1 ]
Ma, Siying [1 ,2 ]
Negre, Nicolas [3 ,4 ]
Gong, Hui [1 ]
White, Kevin P. [3 ,4 ]
Tian, Jingdong [1 ,2 ]
机构
[1] Duke Univ, Dept Biomed Engn, Durham, NC 27706 USA
[2] Duke Univ, Inst Genome Sci & Policy, Durham, NC 27706 USA
[3] Univ Chicago, Inst Genom & Syst Biol, Chicago, IL 60637 USA
[4] Univ Chicago, Dept Human Genet, Chicago, IL 60637 USA
关键词
DNA; AMPLIFICATION; MICROARRAY; CLONING;
D O I
10.1038/nbt.1847
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology(1-3). Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of similar to 0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZ alpha and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.
引用
收藏
页码:449 / +
页数:5
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