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Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq

机译:使用定量术语-SEQ快速生成序列多样化终端库及其参数化

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摘要

Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in Escherichia coli. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common in vivo systems such as E. coli.
机译:合成生物学和合理的设计和建造生物设备的需要特点生物部分的广大,以及可靠的设计工具来构建日益复杂的,多基因结构。对于内在的终结设计原则已经确立;然而,附加的序列 - 结构研究来基于终止遗传设备细化参数。我们报告了快速单罐法生成同时识别终止序列并测量它们的终止效率(TES)的数千名随机双向内在的终结和改进量化期限-SEQ(QTERM-SEQ)方法库。使用QTERM-SEQ,我们表征数百额外强终止子(TE> 90%)与一些终止减弱转录通读在大肠杆菌中高达1000倍。我们终止库和QTERM-Seq的管道构成一个灵活的平台,使的,可以实现转录终止不仅在所需的范围内,而且,调查它们的序列结构特性,包括对超出体内常见的特定遗传和应用程序上下文终止零件识别系统,例如大肠杆菌。

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