EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae.
EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae.
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DOI：
10.1111/1567-1364.12118
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发表时间：
2014-03
影响因子：
3.2
通讯作者：
Borodina I
中科院分区：
文献类型：
--
作者：
Jensen NB;Strucko T;Kildegaard KR;David F;Maury J;Mortensen UH;Forster J;Nielsen J;Borodina I
开发高效生产化学品和药品的菌株需要多轮基因工程。在这项研究中，我们描述了面包酵母酿酒酵母EasyClone载体集的构建和特性，该载体集可以同时表达多个基因，并可以选择回收选择标记。该载体结合了高效的尿嘧啶切除反应克隆和Cre-loxP介导的标记回收系统的优点。通过将编码青色、黄色和红色荧光蛋白的基因插入到单独的载体中，并用流式细胞仪分析蛋白质的共表达，对上体和整合载体集进行了测试。表达来自三个整合的三种荧光蛋白编码基因的细胞显示出比在外体质粒上编码的荧光蛋白的细胞高得多的同时表达水平，相应地，95%和6%的细胞对所有三种颜色的平均荧光区间在log10的荧光区间内。我们证明了利用Cre介导的重组可以同时去除选择性标记，所有整合的异源基因都保留在染色体上，并显示出不变的表达水平。因此，该系统适用于酵母的代谢工程，可以进行多轮基因导入和标记回收。
Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
用户融合：一种快速有效的方法，用于同时融合和克隆多个PCR产品。
影响因子：
14.9
作者：
Geu-Flores, Fernando;Nour-Eldin, Hussam H.;Nielsen, Morten T.;Halkier, Barbara A.
通讯作者：
Halkier, Barbara A.
影响因子：
12.3
作者：
Gentleman RC;Carey VJ;Bates DM;Bolstad B;Dettling M;Dudoit S;Ellis B;Gautier L;Ge Y;Gentry J;Hornik K;Hothorn T;Huber W;Iacus S;Irizarry R;Leisch F;Li C;Maechler M;Rossini AJ;Sawitzki G;Smith C;Smyth G;Tierney L;Yang JY;Zhang J
通讯作者：
Zhang J
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.