Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.
Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.
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DOI：
10.1007/978-1-62703-755-6_8
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发表时间：
2014-02
影响因子：
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通讯作者：
H. Vincent;C. Robinson;Ming-Cheng Wu;N. Dixon;Jason Micklefield
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文献类型：
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作者：
H. Vincent;C. Robinson;Ming-Cheng Wu;N. Dixon;Jason Micklefield
核糖开关是天然存在的基于RNA的遗传开关，其响应于小分子配体的结合而控制基因表达，通常通过调节转录或翻译。它们简单的作用机制和核糖开关类的不断扩大的多样性使它们成为开发新型基因表达工具的有吸引力的靶标。实现这一潜力的第一步是产生对非天然合成配体做出反应的人工核糖开关，从而避免正常细胞功能的破坏。在这里，我们描述了一个战略工程正交选择性核糖开关的基础上天然开关。该方法从天然存在的核糖开关的配体结合口袋的饱和诱变开始，以产生核糖开关突变体的文库。然后针对合成化合物文库在体内筛选这些突变体以鉴定功能性核糖开关-配体组合。有前途的核糖开关配体对，然后在体内和体外进一步表征。使用这种方法，可以产生一系列人工核糖开关，它们是用于蛋白质生产、基因功能分析、代谢工程和其他生物技术应用的通用合成生物学工具。
Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function. Here we describe a strategy for engineering orthogonally selective riboswitches based on natural switches. The approach begins with saturation mutagenesis of the ligand-binding pocket of a naturally occurring riboswitch to generate a library of riboswitch mutants. These mutants are then screened in vivo against a synthetic compound library to identify functional riboswitch–ligand combinations. Promising riboswitch–ligand pairs are then further characterized both in vivo and in vitro. Using this method, a series of artificial riboswitches can be generated that are versatile synthetic biology tools for use in protein production, gene functional analysis, metabolic engineering, and other biotechnological applications.