Dataset of manuscript : "A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications"

T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we developed a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables the scalable design of T7 RNAP based gene regulatory networks and enables the rapid and sensitive detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins. By integrating a protein design pipeline, we generated biosensors using fully synthetic binders, demonstrating the potential for rapid development of novel protein-based sensors. We constructed a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work establishes a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.

T7 RNA聚合酶（T7 RNA polymerase）在合成生物学与生物技术领域应用极为广泛，但对其实现精准且模块化调控的能力仍存在令人意外的局限性。本研究在无细胞系统中构建了一套T7 RNAP调控工具箱，包含可编程的人工合成阻遏蛋白、激活蛋白与生物传感器。该工具箱可支持基于T7 RNAP的基因调控网络的规模化设计，同时能够实现对多种生物分子的快速灵敏检测，涵盖小分子药物、抗体及各类蛋白质。通过整合蛋白质设计流程，本研究利用全合成结合域构建了生物传感器，证明了快速开发新型蛋白质传感器的可行性。我们构建了一套诊断用无细胞系统，整合了SARS-CoV-2刺突蛋白（SARS-CoV-2 Spike protein）感应、基于基因调控的扩增、酶促扩增以及基于葡萄糖的检测模块，展现了高灵敏度即时检测的应用潜力。本研究构建了一套灵活可拓展的基因线路构建框架，可用于构建响应多种生物分子的基因线路，并验证了工程化即时检测无细胞诊断检测方法的应用前景。