Modular protein switches

细胞中成千上万种不同的蛋白质中的每一种都有独特的功能。这些功能通常由其他分子（信号）调节。有些蛋白质是开关;信号分子与蛋白质结合，将其功能从“关闭”状态切换到“打开”状态（反之亦然）。这个开关可以感知到污染物或感染的存在，并触发防御机制。它可以感知癌细胞的存在并触发细胞杀伤反应。潜在的传感和响应能力是巨大的。本项目将开发两个用于创建交换机的模块化平台。一个将检测特定的蛋白质，另一个将控制基因表达。这项提案的研究成功将使其他人能够开发蛋白质开关，并将其应用于生物制造的合成生物学以及基础生物学研究。对本科生和研究生的严格培训将扩大生物技术的劳动力。蛋白质开关识别配体浓度等输入信号，并触发酶活性或DNA结合等输出信号。定向进化已经产生了对它们的配体具有高亲和力和输出变化大的开关。然而，开发合适的选择或筛选来识别开关仍然具有挑战性，并且通常极大地阻碍开关开发。在这个项目中，将开发用于蛋白质传感和基因表达控制的模块化蛋白质开关平台。蛋白质传感平台将利用设计的锚定重复蛋白，并将触发酶活性或荧光。用于基因表达的模块化控制的平台将利用重复调控间隔短回文重复序列（CRISPR）系统的核酸酶无效dCas 9蛋白，以及结合廉价小分子的蛋白质结构域。这些开关将作为基因表达的诱导性阻遏物，而小分子则作为低成本的诱导物。这些模块化平台的开发将为生物技术应用和机械研究提供一套功能开关，以促进未来开关的设计。这些开关将用于基础科研，以了解细胞功能，并在合成生物学和代谢工程的应用研究。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Each of the thousands of different proteins in a cell has a unique function. These functions are often regulated by other molecules (signals). Some proteins are switches; a signal molecule binds to the protein and switches its function from an "off" to an "on" state (or vice versa). The switch could sense the presence of a pollutant, or of an infection, and trigger defense mechanisms. It could sense the presence of cancer cells and trigger a cell-killing response. The potential sensing and response capabilities are tremendous. Two modular platforms for creating switches will be developed during this project. One will detect specific proteins, and the other will control gene expression. Success in the research of this proposal will enable others to develop protein switches with applications in synthetic biology for biomanufacturing as well as in basic biological research. Rigorous training of undergraduates and graduate students will expand the biotechnology workforce.Protein switches recognize input signals such as ligand concentration and trigger an output signal such as enzyme activity or DNA binding. Directed evolution has generated switches with high affinity for their ligand and large changes in output. However, developing suitable selections or screens to identify switches remains challenging and often greatly hinders switch development. In this project, modular protein switch platforms for protein sensing and for control of gene expression will be developed. The protein-sensing platforms will utilize designed ankyrin-repeat proteins and will trigger enzyme activity or fluorescence. The platform for modular control of gene expression will utilize the nuclease-null dCas9 protein of the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system, and protein domains that bind inexpensive small molecules. The switches will function as inducible repressors of gene expression, with the small molecules serving as low-cost inducers. The development of these modular platforms will provide a set of functional switches for biotechnological use and for mechanistic study to facilitate the design of future switches. These switches will be used in basic scientific research to understand cellular function and applied research in synthetic biology and metabolic engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.