A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences.
A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences.
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DOI：
10.1186/1475-2859-12-47
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发表时间：
2013-05-10
影响因子：
6.4
通讯作者：
Daran-Lapujade P
中科院分区：
文献类型：
--
作者：
Kuijpers NG;Solis-Escalante D;Bosman L;van den Broek M;Pronk JT;Daran JM;Daran-Lapujade P
用于在酿酒酵母中组装大 DNA 构建体的重叠 DNA 片段的体内重组对于小实验室规模的途径工程以及自动化高通量菌株构建具有巨大的潜力。然而，目前的体内组装方法在正确组装结构的产量方面并不一致，并且尚未探索常规实验室实施所需部件的标准化。在这里，我们提出并评估了一种优化且稳健的方法，用于在酿酒酵母中从重叠 DNA 片段体内组装质粒。为了最大限度地减少错误组装质粒的发生并增加组装平台的多功能性，引入了两项主要改进； i) 载体骨架的基本元件（酵母附加体和选择标记）被断开，ii) 在组装片段的每个侧翼引入与酵母基因组非同源的标准化 60 bp 合成重组序列。与之前的方法相比，这些修改导致源自主干的假阳性转化体减少了 100 倍。 60 bp 合成重组序列的实施使复杂表达构建体的设计具有高度灵活性，并允许通过 PCR 快速轻松地构建所有组装片段。该方法的功能通过从携带六个糖酵解基因的九个重叠片段中组装出 21 kb 质粒来证明，正确组装率为 95%。组装的质粒被证明是计算机设计的高保真复制品，并且质粒携带的所有糖酵解基因都被证明是有功能的。所提出的方法为酿酒酵母中多片段表达载体的组装提供了实质性改进。它不仅提高了体内组装的效率，而且还提供了一个多功能平台，可以轻松快速地设计和组装合成结构。因此，所提出的方法非常适合复杂途径的构建和用于代谢工程目的的高通量菌株构建程序。此外，其稳健性和易用性有利于构建任何携带两个或多个基因的质粒。
In vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae. To minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional. The presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitate the construction of any plasmid carrying two or more genes.
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影响因子：
14.9
作者：
Gibson DG
通讯作者：
Gibson DG
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
14.9
作者：
MANIVASAKAM, P;WEBER, SC;SCHIESTL, RH
通讯作者：
SCHIESTL, RH
影响因子：
2.6
作者：
Campagno, C;Brambilla, L;Porro, D
通讯作者：
Porro, D
影响因子：
2.7
作者：
Raymond, CK;Pownder, TA;Sexson, SL
通讯作者：
Sexson, SL