Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting,ACS Synthetic Biology

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Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-08-17 00:00:00 , DOI: 10.1021/acssynbio.8b00099
Katharine Page ₁ , Jeremy Shaffer ₁ , Samuel Lin ₁ , Mark Zhang ₁ , Jane M. Liu ₁

Affiliation

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 10⁸ random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N₄₀ sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3–5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3–5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

中文翻译：

使用双重遗传选择和荧光激活细胞分选技术体内工程Riboswitchs

核糖开关是结合小分子效应子以在转录或翻译水平上控制基因表达的非编码RNA，特别适合应对合成生物学中的挑战。为了扩展现有核糖开关的有限集，我们开发了一个核糖开关发现平台，该平台结合了双重遗传选择和荧光激活的细胞分选，以从10 ^8个随机序列文库中鉴定出新型的核糖开关，其中ThiM＃2核糖开关的适体结构域是换成N ₄₀顺序。在原理验证中，我们确定了小分子茶碱的新型核糖开关。我们最好的核糖开关（命中率3-5）在茶碱存在下显示下游基因表达的2.3倍活化。Hit 3-5的随机诱变，加上选择和筛选，提供了改良的核糖开关，显示了近3倍的激活。据我们所知，这是适体结构域在体内定向进化以生成功能性核糖开关的第一个报道。

更新日期：2018-08-17

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