Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System
Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System
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DOI：
10.1021/acssynbio.7b00322
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发表时间：
2018-03-01
影响因子：
4.7
通讯作者：
Moon, Tae Seok
中科院分区：
文献类型：
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作者：
Lee, Young Je;Kim, Soo-Jung;Moon, Tae Seok
合成小 RNA 调节剂已成为可预测控制细菌基因表达的多功能工具。由于其简单的设计原理、小尺寸和高度正交的行为，这些工程遗传部分已被纳入遗传电路中。然而，由于我们将不同 RNA 调节因子整合到复杂电路中的能力有限，利用 RNA 调节因子实现更复杂的细胞功能的努力受到了阻碍。在这里，我们提出了大肠杆菌中的组合 RNA 调控系统，该系统使用小转录激活 RNA (STAR) 和反义 RNA (asRNA) 以可编程方式激活或失活靶基因表达。具体来说，我们证明了 STAR 系统激活的靶标输出可以通过表达两种不同类型的 asRNA 来失活：一种与 STAR 调节因子结合并隔离，影响转录过程，而另一种与靶标 mRNA 结合，影响翻译过程。我们通过优化每种类型的 asRNA，然后将两种优化的 asRNA 集成到单个电路中，提高了失活效率（高达 96%）。此外，我们证明了组合的STAR和asRNA系统可以以可逆的方式控制基因表达，并且可以调节基因组中基因的表达。最后，我们在同一单元中构建并同时测试了两个 A AND NOT B 逻辑门，以显示组合系统复杂的多基因调控。我们的方法建立了一种整合多个 RNA 调节因子以合理控制多个基因的方法。
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.