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Chemoenzymatic construction of a programmable synthetic endoplasmic reticulum

可编程合成内质网的化学酶构建

基本信息

批准号：
2124105
负责人：
Neal Devaraj
金额：
$ 150万
依托单位：
University of California-San Diego
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2124105&HistoricalAwards=false
关键词：
Chemoenzymatic construction programmable synthetic endoplasmic

项目摘要

A fundamental goal in synthetic biology is to build artificial cells that have many of the basic functions of natural cells, as well as specific functions that are useful in medicine and biotechnology. This project focuses on building a synthetic subcellular compartment or organelle known as the endoplasmic reticulum, since this organelle would provide artificial cells with essential functions such as the ability to synthesize proteins, process proteins and synthesize lipids. Broader outcomes of this proposal include graduate student training and educating the public about designer cell organelles and their importance through outreach lectures to high school students and the general public. Bioethical consequences related to generating a synthetic organelle will also be considered. To generate a synthetic ER de novo, the chemoenzymatic synthesis of non-canonical phospholipids will be combined with recombinant proteins that influence membrane curvature and fusion. The proposed program to generate and study a synthetic ER will follow these three closely coupled objectives: (1) chemical and enzymatic steps will be developed to generate a library of non-canonical phospholipid membranes similar to the lipid membranes of living cells. (2) the minimal lipid composition requirements necessary for generating an ER-like reticulated membrane architecture will be explored. The interactions of these lipids with membrane-bound proteins that play a critical role in membrane curvature stabilization and fusion respectively in yeast, will be monitored. Furthermore, different combinations of non-canonical lipids will be tested to explore how biomimetic phospholipids affect membrane properties; and (3) the differences in properties between natural and synthetic spherical, tubular, and reticular membrane geometries will be investigated. In particular, the kinetics of internalized reactions, reversibility of the steady-state morphology, mixing of lipids in reticular membranes, diffusion through the lumen of the synthetic ER, and network connectivity will be characterized. To equip the synthetic ER with functionality, a simple post-translational network by localizing two fluorescently labeled glycosyltransferases will be developed within a synthetic reticular network for post-translational modification of a protein substrate. The proposed studies will establish the groundwork for developing artificial reticulated organelles that mimic the ER and facilitate future programmable functions such as post-translational modification, biomolecular trafficking, sensing, and protein turnover.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.

合成生物学的一个基本目标是构建具有天然细胞的许多基本功能以及在医学和生物技术中有用的特定功能的人工细胞。该项目的重点是建立一个被称为内质网的合成亚细胞区室或细胞器，因为这个细胞器将为人工细胞提供必要的功能，如合成蛋白质，加工蛋白质和合成脂质的能力。这一建议的更广泛成果包括研究生培训和教育公众关于设计师细胞器及其重要性，通过对高中生和公众的外联讲座。还将考虑与产生合成细胞器有关的生物伦理后果。为了从头产生合成ER，将非典型磷脂的化学酶促合成与影响膜曲率和融合的重组蛋白质组合。所提出的产生和研究合成ER的计划将遵循这三个紧密耦合的目标：（1）将开发化学和酶促步骤以产生类似于活细胞的脂质膜的非规范磷脂膜的文库。(2)将探索产生ER样网状膜结构所需的最低脂质组成要求。将监测这些脂质与膜结合蛋白的相互作用，所述膜结合蛋白分别在酵母中的膜曲率稳定和融合中起关键作用。此外，将测试非典型脂质的不同组合以探索仿生磷脂如何影响膜性质;以及（3）将研究天然和合成的球形、管状和网状膜几何形状之间的性质差异。特别是，内化反应的动力学，稳态形态的可逆性，脂质在网状膜中的混合，通过合成ER的管腔的扩散，和网络连接将被表征。为了使合成ER具有功能性，将在合成网状网络内通过定位两种荧光标记的糖基转移酶来开发简单的翻译后网络，用于蛋白质底物的翻译后修饰。这项研究将为开发人工网状细胞器奠定基础，这些细胞器模仿ER并促进未来的可编程功能，如翻译后修饰，生物分子运输，传感和蛋白质周转。该奖项反映了NSF的法定使命，并被认为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来支持。