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DNA assembler. 韩世磊. 通过 DNA assembler 进行天然产物生物合成途径的快速构建和工程化. 前言. 在途径工程和代谢工程当中,常常将原产生菌中的完整的生物合成途径体外克隆转移到 异源有机体 中进行表达,以获得一定产量的产品。 传统的多步连续的克隆方法,包括限制性酶切,体外连接和转化,需要很多的质粒载体,这些方法不仅耗时而且效率也不高,并且依赖于特异性的限制性酶切位点,这限制了大规模的 DNA 分子的重组。

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Dna assembler

DNA assembler


通过DNA assembler进行天然产物生物合成途径的快速构建和工程化




由于酵母同源重组容易进行而且高效,因此在酵母中进行同源重组已经被广泛的应用于基因克隆,质粒构建和文库构建。在这里,本文献介绍了一种新的方法,叫DNA assembler。它依赖酵母细胞中的同源重组机制,设计并且快速的构建大规模的生化途径,通过一步即可完成。




  • overlaps of 400bp between internal adiacent pathway fragments引物不需要尾巴不必太长

  • overlaps of 40bp between the last pathway fragments and the S.cerevisiae helper fragment.


  • overlaps of 80bp between the first pathway fragments and the S.lividans.

  • two adjacent gene expression cassettes. overlap extension PCR (OE-PCR).

  • fused (成环)through in vivo homology recombination (HR)

  • To 50 bp, the reverse primer used to amplify Tx contains a sequence of the first 20-25 nucleotides of Px+1, and the forward primer used to amplify Px+1 contains a sequence of the last 20-25 nucleotides of Tx;

  • to 125 bp ;100 nucleotides of Tx after which it was joined

  • To 270-430 bp, Tx was fused with Px+1, Gx+1 and Tx+1 through OE-PCR.


  • 7个平均大小为4-5KB的途径片段(共29.1kb)

  • 3个大肠、链霉、酵母的辅助片段(共6.6KB)

  • 一共35.7KB的各个片段共转化进酵母中。

  • CEN6ARS H4酵母辅助片段中的复制子

  • Ura3 ----选择标记

  • oriR6K大肠复制子

  • accIV ---编码安普霉素抗性

  • oriT-----结合转移起始位点

Dna assembler1
DNA assembler重要材料:

Plasmids pAE4 Streptomyces - E. coli shuttle vector pAE4 (扩增大肠杆菌和链霉菌辅助片段)

The plasmid pRS416 and pRS426(扩增酵母辅助片段)

Synthetic complete drop-out medium lacking ura (SC-Ura) was used to select transformation.

Saccharomyces cerevisiae HZ848 (MATα, ade2-1, Δura3, his3-11, 15, trp1-1, leu2-3, 112, and can 1-100)

S. cerevisiae YSG50 (MATa, ade2-1, ade322, ura3-1, his3-11,15, trp1-1, leu2-3,112 and can1-100)


Pathway fragments were amplified from the genomic DNA.

The S.cerevisiae helper fragment pRS416, the E.coli helper fragment and the S.lividans helper fragment pAE4

200-300ng of each individual fragment was mixed and precipitated with ethanol.

The resulting DNA pellet was air-dried and resuspended in 4 µL Milli-Q double deionized water去离子水。

After being concentrated, the mixture was electroporatedinto S.cerevisiae or transformed to S.cerevisiae using the lithium acetate锂乙酸盐/single stranded carrier DNA/polyethylene glycol (PEG) method.


Colonies were randomly picked to SC-Ura liquid media and grown for one day, after which the plasmids from yeast

Yeast plasmids were transformed to E.coli strain BW25141 and selected on Luria Broth(LB) agar plates(apramycin)

plasmids were isolated from the liquid culture Plasmids isolated from E. coli were then subjected to restriction digestion.

Dna assembler2
DNA assembler特点

SLIC (Sesequence and ligation-independent cloning) 成功率很低only 17%(7 of 42 E.coli transformants are proven to be correct),重组片段较小,仅仅8KB左右。Li,M.Z. and Elledge, S.J. (2007) Harnessing homologous

需要T4 DNA Polymerase and RecA recombinase 在转化之前对DNA片段进行处理。Homologous recombination 在酵母中的同源重组比在细菌中以及其他高等的真核生物中的效率更高。


Dna assembler3
DNA assembler特点

domino clones’ in a vector through in vivo homologous recombination 需要首先制备载体,组装过程顺序进行,费时费力。

a,M., Fujita,K., Kuroki,A. and Tsuge,K. (2008)

而DNA Assembler 快速、高效、可以构建大的重组DNA分子



Dna assembler4
DNA assembler特点






Zengyi shao a yunzi luo b and huimin zhao
作者简介Zengyi shaoa Yunzi Luob and Huimin Zhao※