RoL: EAGER: DESYN-C3: Moving information across synthetic membranes via engineered sensors

RoL：EAGER：DESYN-C3：通过工程传感器跨合成膜移动信息

• 批准号: 1844219
• 负责人: Neha Kamat
• 金额: $ 30万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1844219&HistoricalAwards=false
• 关键词: RoL; EAGER; DESYN; C3; Moving

Pseudocell engineering is the design and synthesis of structures that mimic real cell functions. These structures can be useful for biotechnological applications or to advance our understanding of cellular processes. Cell membranes often isolate intracellular reactions from the extracellular environment. To enable pseudocells to control when and how reactions take place requires technologies to sense the external environment without requiring a flow of materials. Living cells accomplish this goal using sensor proteins installed within the cell's outer membrane. This project will coordinately design proteins and membranes to perform similar sensory and signaling roles. This project will also train students and will enhance ongoing outreach activities. A diverse STEM workforce will be developed by engaging undergraduates in research through Northwestern University's iGEM team and its synthetic biology-focused REU, SynBREU.This project will develop strategies for embedding protein sensors in synthetic membranes. This will help uncover relationships between membrane and protein design features. Achieving productive biosensor performance will be integral to evaluating the design process. The approach taken is based on preliminary data that demonstrates the feasibility of using cell-free expression systems to insert a functional channel protein (MscL) into vesicle membranes. This will be combined with the Modular Extracellular Sensing Architecture (MESA) developed by the co-PI to generate novel biosensors that are orthogonal to native systems. Ligand binding induces dimerization of MESA chains triggering an internal response without transmembrane transport of material. This approach will be evaluated using rapamycin as the external signal and split green fluorescent protein as the internal reporter. If successful, the approach will be extended to using vascular endothelial growth factor as the signal molecule and split Nano-Luciferase as the reporter. If successful, this project will benefit society by ultimately enabling the use of pseudocell technology for improved environmental stewardship, and for improved patient care and treatment. The technologies to be developed here will benefit the biotechnology and pharmaceutical industries by enabling the development of new classes of therapeutic and diagnostic products.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.

假细胞工程是模拟真实的细胞功能的结构的设计和合成。 这些结构可以用于生物技术应用或促进我们对细胞过程的理解。细胞膜通常将细胞内反应与细胞外环境隔离。为了使假细胞能够控制反应何时以及如何发生，需要在不需要材料流动的情况下感知外部环境的技术。活细胞利用安装在细胞外膜内的传感器蛋白来实现这一目标。该项目将协调设计蛋白质和膜，以执行类似的感觉和信号作用。该项目还将培训学生，并将加强正在进行的外联活动。 通过西北大学的iGEM团队及其以合成生物学为重点的REU，SynBREU，将通过让本科生参与研究来培养多样化的STEM劳动力。该项目将开发在合成膜中嵌入蛋白质传感器的策略。这将有助于揭示膜和蛋白质设计特征之间的关系。实现生产性生物传感器性能将是评估设计过程不可或缺的。所采取的方法是基于初步的数据，证明了使用无细胞表达系统的可行性插入到囊泡膜的功能通道蛋白（MscL）。这将与共同PI开发的模块化细胞外传感架构（MESA）相结合，以生成与天然系统正交的新型生物传感器。配体结合诱导MESA链的二聚化，触发内部反应，而没有物质的跨膜转运。该方法将使用雷帕霉素作为外部信号并使用分裂的绿色荧光蛋白作为内部报告物进行评价。如果成功，该方法将扩展到使用血管内皮生长因子作为信号分子和分裂纳米荧光素酶作为报告分子。 如果成功，该项目将通过最终使假细胞技术用于改善环境管理以及改善患者护理和治疗而造福社会。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer

将跨膜机械敏感通道蛋白无细胞合成到混合支持的脂质双层中

• DOI: 10.1021/acsabm.0c01482
• 发表时间: 2021
• 期刊: ACS Applied Bio Materials
• 影响因子: 4.7
• 作者: Manzer, Zachary A.;Ghosh, Surajit;Jacobs, Miranda L.;Krishnan, Srinivasan;Zipfel, Warren R.;Piñeros, Miguel;Kamat, Neha P.;Daniel, Susan
• 通讯作者: Daniel, Susan

Vesicle-Based Sensors for Extracellular Potassium Detection

• DOI: 10.1007/s12195-021-00688-7
• 发表时间: 2021-08-10
• 期刊: CELLULAR AND MOLECULAR BIOENGINEERING
• 影响因子: 2.8
• 作者: Boyd, Margrethe A.;Davis, Anna M.;Kamat, Neha P.
• 通讯作者: Kamat, Neha P.