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HomeRun Vector Assembly System: A Flexible and Standardized Cloning System for Assembly of Multi-Modular DNA Constructs

机译：HomeRun载体组装系统：用于组装多模块DNA构建体的灵活标准化克隆系统

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Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector Assembly System (HVAS), employs a three-tiered vector series that utilizes both multisite gateway cloning and homing endonucleases, with the former building individual functional modules and the latter linking modules into the final construct. As a proof-of-principle, we first built a two-module construct that supported doxycycline-induced expression of green fluorescent protein (GFP). Further, with a three-module construct we showed quantitatively that there was minimal promoter leakage between neighbouring modules. Finally, we developed a method, in vitro Cre recombinase-mediated cassette exchange (RMCE) cloning, to regenerate a gateway destination vector from a previous multisite gateway cloning reaction, allowing access to existing DNA element libraries in conventional gateway entry clones, and simple creation of constructs ready for in vivo RMCE. We believe these methods constitute a useful addition to the standard molecular cloning techniques that could potentially support industrial scale synthesis of DNA constructs.
机译：分子生物学和合成生物学的进步要求有效组装多模块DNA构建体。我们在此提出一种新颖的模块化克隆方法，该方法消除了对限制性核酸内切酶的需求，并通过标准化所有DNA元件和克隆反应，显着提高了复杂DNA分子的设计和构建效率。我们的系统称为HomeRun载体装配系统（HVAS），它采用三层载体系列，该系列利用多站点网关克隆和归巢内切核酸酶，前者构建单个功能模块，后者将模块链接到最终构建体中。作为原理的证明，我们首先构建了一个支持多西环素诱导的绿色荧光蛋白（GFP）表达的两模块构建体。此外，使用三模块构建体，我们定量显示相邻模块之间的启动子泄漏最少。最后，我们开发了一种方法，通过体外Cre重组酶介导的盒式交换（RMCE）克隆，从以前的多站点网关克隆反应中重新生成网关目标载体，从而可以访问常规网关条目克隆中的现有DNA元件库，并且可以轻松创建准备用于体内RMCE的构建体。我们认为这些方法构成了对标准分子克隆技术的有用补充，可以潜在地支持工业规模合成DNA构建体。

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