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首页> 外文期刊>Applied Microbiology and Biotechnology >Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property
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Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property

机译：Theophylline Riboswitch的综合表征揭示了Shine-Dalgarno影响活性翻译性的两个枢轴特征

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摘要

Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.
机译：由合成生物元素控制的可调基因表达对生物技术和合成生物学非常重要。合成核素是一种枢转类型的元素，可以容易地控制各种细菌中的异源基因表达。在该研究中，采用了依赖性依赖性合成核糖干线和光照 - 达尔加诺（SD）序列和起始密码子之间的变化的相应变体来全面地表征诱导和调节性能，通过在枯草芽孢杆菌中组合强大的启动子APRE。在新构造的表达元件集中，具有9-BP间隔的表达元件表现出更高的表达水平，优异的感应倍率，并且比具有较长或更短的间距的泄漏表达相当较低。具有9-BP间隔的核糖开关表达元件显示出大致线性剂量依赖性从0到8mm的茶碱。通过在天然序列之前通过插入单个基座的修改SD序列使得能够增加表达水平后的诱导，同时由于泄漏水平升高而降低感应折叠。具有工程化SD和最佳9-BP间距的核糖开关元件表现出改变的剂量依赖性，其中大致线性范围偏移到0-4mm，但它具有与感应过程类似的轮廓。这些结果不仅为茶碱核素相关的诱导表达提供了来自B.枯草芽孢杆菌的强烈天然启动子的常识数据，而且为其他合成核糖开关的模块化设计提供了对SD的两个枢转特征。

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