Dna assembler
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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

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Dna assembler

DNA assembler

韩世磊


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


Dna assembler

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。

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。


Dna assembler

方法


Dna assembler

酵母


Dna assembler

酵母

大肠

链霉


Dna assembler

  • 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.

    用带有尾巴的引物扩增酵母辅助片段的时候遇到困难,因此最后用不带尾巴的引物进行PCR。

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


Dna assembler

  • 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.


Dna assembler

金链霉素

  • 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。


Dna assembler

  • 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)


Dna assembler

操作过程

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.


Dna assembler

结果鉴定

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 assembler

总结


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。

仅仅是利用酵母细胞内本就存在的同源重组机制,因此不需要任何外源的重组酶。仅仅需要通过PCR制备DNA样品,以及一步转化。


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

与常规基因质粒构建不同,可以一步组装多基因生化途径

可以构建特定的DNA分子,组装到质粒上或者在染色体上(使外源DNA稳定的存在于细胞内,并且有组装大的生化途径或者整个基因组的潜能)。


Dna assembler4

DNA assembler特点

进行定点突变

传统的方法费时费力,用这种方法仅仅需要在扩增途径片段进行引物设计的时候,将点突变设计到引物中去。

通过引物设计进行基因的缺失


Dna assembler

谢谢

2012—04--11


Zengyi shao a yunzi luo b and huimin zhao

作者简介Zengyi shaoa Yunzi Luob and Huimin Zhao※


Dna assembler

两步组装法


Dna assembler

参考文献


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