Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea.
Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
Biotechnol J. 2022 Mar;17(3):e2000449. doi: 10.1002/biot.202000449. Epub 2021 Apr 29.
Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. We first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks in vitro. As a step to quickly assess the feasibility of aptamer functions in vivo, we tested the aptamer and its sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. The expression of aptamer in E. coli demonstrated control over GFP expression driven by T7 RNA polymerase, indicating its ability to serve as building blocks for logic circuits and transcriptional cascades. This work elucidates the potential of T7 RNA polymerase aptamer as regulators for synthetic biological circuits and metabolic engineering.
近年来，核酸工程的进展引入了几种基于 RNA 的调控元件，用于合成基因回路，从而扩展了工程生物体的工具集。在这项工作中，我们设计了遗传电路，实现了先前描述的能够与 T7 RNA 聚合酶结合并在体外抑制其活性的 RNA 适体。我们首先证明了 RNA 适体与可编程合成转录网络结合在体外的实用性。作为快速评估适体在体内功能可行性的一步，我们在无细胞表达系统中测试了适体及其序列变体，验证了适体在无细胞实验中的功能。在大肠杆菌中表达适体可控制由 T7 RNA 聚合酶驱动的 GFP 表达，表明其有能力作为逻辑电路和转录级联的构建块。这项工作阐明了 T7 RNA 聚合酶适体作为合成生物回路和代谢工程调节剂的潜力。
