A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae
A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae
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DOI：
10.1002/yea.3144
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发表时间：
2016-03-01
期刊：
影响因子：
2.6
通讯作者：
Rudolf, Fabian
中科院分区：
文献类型：
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作者：
Gnuegge, Robert;Liphardt, Thomas;Rudolf, Fabian
穿梭载体允许将重组DNA有效转移到酵母细胞中，并广泛用于基础研究和生物技术。虽然可用的穿梭载体适用于许多实验环境，但它们在定量生物学中的使用受到不充分的拷贝数控制的阻碍。此外，它们通常具有实际限制，例如有限的模块化和很少的独特限制位点。我们构建了pRG穿梭载体系列，由单拷贝和多拷贝整合型、着丝粒型和附加型质粒组成，这些质粒具有用于在所有常用的营养缺陷型酵母菌株中进行选择的标记基因。这些载体具有模块化设计和大量独特的限制性位点，能够有效交换每个载体部分并扩展系列。使用双交换重组机制实现整合到宿主基因组中，从而产生稳定的单拷贝和多拷贝修饰。由于着丝粒和附加型质粒产生异质细胞群，因此对其拷贝数分布和丢失行为进行了分析。总的来说，穿梭载体系列支持基因的有效克隆及其在酵母细胞中的维持，并改善了拷贝数控制。版权所有（C）2015约翰威利父子有限公司
Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single- and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control. Copyright (C) 2015 John Wiley & Sons, Ltd.