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Single Day Construction of Multigene Circuits with 3G Assembly
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2018-05-01 00:00:00 , DOI: 10.1021/acssynbio.8b00060
Andrew D. Halleran , Anandh Swaminathan , Richard M. Murray

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly’s ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.

中文翻译:

具有3G组件的多基因电路的单日构建

快速设计,构建和测试原型的能力对于每个工程学科都至关重要。DNA装配通常是合成生物学原型周期的限速步骤。最近开发的DNA组装方法(如等温组装和IIS型限制酶系统)采用了不同的方法来加速DNA的构建。我们介绍了一种混合方法,Golden Gate-Gibson(3G),该方法利用了由IIS型限制酶系统引入的模块式零件库以及等温装配的能力,可在单罐反应中构建大型DNA构建体。我们的方法高效,快速,可在一天内完成整个多基因电路的构建。此外,3G允许生成变体库,从而可以有效筛选不同的可能电路结构。
更新日期:2018-05-01
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