Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York.
Department of Mechanical Engineering, University of California at Riverside, Riverside, California.
Biotechnol Bioeng. 2019 May;116(5):1139-1151. doi: 10.1002/bit.26918. Epub 2019 Feb 4.
To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.
为了构建具有可预测行为的复杂遗传网络，合成生物学家使用模块化部件库，这些部件可以在分离状态下进行特征描述，并组合在一起，以创建可编程的更高阶功能。用于单独操作的基因调控部件的特征实验和计算模型通常用于预测相互连接的部件的动态，并指导新的合成器件的构建。在这里，我们分别使用小转录激活 RNA（STARs）和簇状规律间隔短回文重复干扰（CRISPRi）来表征两种基于 RNA 的转录调控模式，并展示它们不同的调控时间尺度如何用于设计组成的前馈回路，从而产生基因表达的脉冲。我们使用无细胞转录 - 翻译系统（TXTL）来快速表征该系统，并应用贝叶斯推断从基于常微分方程的机械模型中提取动力学参数。然后，我们在模拟中进行演示，并通过 TXTL 实验验证，STARs 和 CRISPRi 同时对单个基因靶标进行调控会导致基因表达的脉冲。我们的结果表明，两种调节剂在集成遗传回路中的模块化，并且我们预计随着合成回路的复杂性增加，能够利用这种模块化的构建和建模框架将变得越来越重要。
