Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System,ACS Synthetic Biology

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Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-02-11 00:00:00 , DOI: 10.1021/acssynbio.7b00322
Young Je Lee ₁ , Soo-Jung Kim ₁ , Tae Seok Moon ₁

Affiliation

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

中文翻译：

STAR和反义RNA组合系统对细菌基因表达的多级调节

合成的小RNA调节剂已成为一种可预测地控制细菌基因表达的多功能工具。由于其简单的设计原理，小巧的尺寸和高度正交的行为，这些基因工程部件已被整合到遗传电路中。但是，由于我们将不同的RNA调节剂整合到复杂电路中的能力有限，因此阻碍了使用RNA调节剂实现更复杂的细胞功能的努力。在这里，我们介绍了大肠杆菌中的组合RNA调节系统它使用小转录激活RNA（STAR）和反义RNA（asRNA）以可编程方式激活或失活靶基因表达。具体而言，我们证明了通过表达两种不同类型的asRNA可以使STAR系统激活的靶标输出失活：一种与as结合并螯合STAR调节剂，影响转录过程，而另一种与靶标mRNA结合，影响翻译。过程。通过优化每种类型的asRNA，然后将两种优化的asRNA整合到单个电路中，我们提高了失活效率（高达96％）。此外，我们证明了STAR和asRNA组合系统可以以可逆的方式控制基因表达，并可以调节基因在基因组中的表达。最后，我们在同一个单元中构建并同时测试了两个A AND NOT B逻辑门，以显示通过组合系统进行的复杂多基因调节。我们的方法建立了整合多个RNA调节剂以合理控制多个基因的方法。

更新日期：2018-02-11

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