Rajkumar Arun S, Maerkl Sebastian J
School of Engineering, Institute of Bioengineering, École Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
ACS Synth Biol. 2012 Oct 19;1(10):483-90. doi: 10.1021/sb300045j. Epub 2012 Jun 20.
Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. Overall, 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions and may serve as tunable components for synthetic genetic networks.
当前的基因合成方法能够生成双链DNA的长片段。我们证明这些技术可用于创建合成调控元件，并描述了一种从酿酒酵母（Saccharomyces cerevisae）中创建完全确定的PHO5启动子合成变体的方法。总体而言，通过高温连接组装了128个启动子，通过等温组装克隆到质粒中，保存在大肠杆菌中，随后通过同源重组转化到酵母中。合成错误出现的频率与当前基因合成方法相当或更低。这里报道的启动子合成方法稳健、快速且易于操作。合成工程化的启动子文库将成为剖析启动子输入-输出功能复杂性的有用工具，并可作为合成遗传网络的可调组件。
