Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
Interfaculty Doctoral Study of Biomedicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
ACS Synth Biol. 2020 Aug 21;9(8):2055-2065. doi: 10.1021/acssynbio.0c00133. Epub 2020 Jul 20.
An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.
合成生物学电路的一个重要特点是它们对外界化学物质信号的响应，这使得细胞过程可以得到外部控制。钙依赖性调节是实现这种控制的一种有吸引力的方法，因为各种刺激通过激活膜通道受体诱导钙离子内流。大多数钙依赖性基因回路都使用内源性激活 T 细胞的核因子（NFAT）信号通路。在这里，我们利用工程化的 NFAT 转录因子诱导 HEK293T 细胞中外源基因表达的强烈和稳健激活。此外，我们设计了一种不干扰 NFAT 调节启动子的钙依赖性转录因子，能够在几种哺乳动物细胞类型中强力激活转录。此外，我们证明将该电路与钙选择性离子通道偶联可导致辣椒素和温度控制的基因表达。这种工程化的钙依赖性信号通路可通过不同的刺激在哺乳动物细胞中实现对基因表达的紧密控制，并且具有多功能性、适应性，可广泛应用于治疗和诊断应用。
