Hwang Yunhee, Kim Seong Gyeong, Jang Sungho, Kim Jongmin, Jung Gyoo Yeol
Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk, 37673, South Korea.
Department of Bioengineering and Nano-Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon, 22012, South Korea.
J Biol Eng. 2021 Mar 19;15(1):11. doi: 10.1186/s13036-021-00261-w.
Synthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive sensor modules for use in synthetic biology applications. However, tuning the fold-change of riboswitch circuits is challenging because a deep understanding of the riboswitch mechanism and screening of mutant libraries is generally required. Therefore, novel molecular parts and strategies for straightforward tuning of the fold-change of riboswitch circuits are needed.
In this study, we devised a toehold switch-based modulator approach that combines a hybrid input construct consisting of a riboswitch and transcriptional repressor and de-novo-designed riboregulators named toehold switches. First, the introduction of a pair of toehold switches and triggers as a downstream signal-processing module to the hybrid input for coenzyme B resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on balancing the expression levels of the RNA components greatly improved the fold-change from 260- to 887-fold depending on the promoter and host strain. Further characterizations confirmed low leakiness and high orthogonality of five toehold switch pairs, indicating the broad applicability of this strategy to riboswitch tuning.
The toehold switch-based modulator substantially improved the fold-change compared to the previous sensors with only the hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.
合成生物电路被广泛用于控制微生物细胞功能。天然和合成核糖开关是合成生物学应用中颇具吸引力的传感器模块。然而，调节核糖开关电路的倍数变化具有挑战性，因为通常需要深入了解核糖开关机制并筛选突变文库。因此，需要新的分子元件和策略来直接调节核糖开关电路的倍数变化。
在本研究中，我们设计了一种基于toehold开关的调节方法，该方法将由核糖开关和转录阻遏物组成的混合输入构建体与名为toehold开关的从头设计的核糖调节因子相结合。首先，将一对toehold开关和触发物作为下游信号处理模块引入到辅酶B的混合输入中，形成了一个功能性核糖开关电路。接下来，几种侧重于平衡RNA组分表达水平的优化策略极大地提高了倍数变化，根据启动子和宿主菌株的不同，倍数变化从260倍提高到887倍。进一步的表征证实了五个toehold开关对的低泄漏性和高正交性，表明该策略在核糖开关调节方面具有广泛的适用性。
与仅具有混合输入构建体的先前传感器相比，基于toehold开关的调节剂显著提高了倍数变化。适合计算机设计和优化的可编程RNA-RNA相互作用可以促进基于RNA的遗传调节剂的进一步开发，以灵活调节核糖开关电路和合成生物传感器。
