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Journal|[J]ACS Synthetic BiologyVolume 5, Issue 4. 2016. PP 344-55
MT全大肠杆菌TX - TL Toolbox 2.0:无细胞合成生物学平台。
The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.
摘要 / Abstract
我们报告并提供了最近开发的所有大肠杆菌,无细胞转录和翻译系统的性能和特性的详细表征。基因表达完全基于大肠杆菌细胞质提取物提供的内源性翻译成分和转录机制,从而将调节部分的目录扩展到数百种元素。我们利用ATP再生的强大代谢能力,在间歇模式反应中实现了2 mg / m L以上的蛋白质合成,在半连续模式中实现了6 mg / m L以上的蛋白质合成。虽然无细胞表达的强度平均增加了3倍,但与以前的结果相比,简单基因线路的输出信号和整个噬菌体的合成都增加了数量级。利用大肠杆菌Maz F干扰酶和Clp XP AAA蛋白酶分别调节信使RNA和蛋白质降解,其速率范围比第一个版本的无细胞工具箱要广泛得多。这个系统是一个高度通用的无细胞平台,通过执行由合成和天然细菌调节部分组成的DNA程序来构建复杂的生物系统。
We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
关键词 / Keywords
生物合成; 无细胞转录-翻译; 基因线路原型化; 最小细胞; 自组装
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