RoL:EAGER:DESYN-C3 Programmable Porous Lipid Sponges as Synthetic Cell Factories

有了这个奖项，化学部的生命过程化学计划，作为生命规则（RoL）的一部分：合成细胞和细胞组分的设计和工程（DESYN-C3）倡议，正在资助来自圣地亚哥加州大学的Neal Devaraj博士，研究脂质“海绵”作为可编程隔室应用于合成细胞的设计。这项工作利用了Devaraj实验室的一个令人兴奋和意想不到的发现，即仿生材料可以被编程为从溶液中吸收生物分子，并将它们集中在海绵状的内部。合成细胞有望通过克服活细胞所面临的固有限制（例如它们无法承受恶劣条件和毒素）来彻底改变生物制造。与人体器官相似，活细胞已经进化为具有称为细胞器的内部隔室，其具有特定功能，并通过浓缩反应物或分离相互不相容的反应来帮助细胞维持和发挥功能。然而，目前还不清楚有多少空间组织和区室化是必要的建设一个合成细胞。脂质海绵可以帮助回答这个问题，因为它们可以被编程以捕获不同类别的生物分子和反应。本计画培养超分子化学、生物化学、软物质及分子生物学之研究生。这项工作还有助于推广活动，将合成细胞的概念引入更广泛的圣地亚哥教育界，旨在刺激低收入和代表性不足的学生群体进入STEM领域。这些研究提供了独特的见解，以了解区室化如何帮助控制生命的复杂化学反应。 活细胞具有高得惊人的大分子浓度。限制和拥挤效应在基因表达、蛋白质折叠和酶促反应的动力学中起着关键作用。在传统的合成细胞模型如囊泡内实现可再现的高浓度大分子是极具挑战性的。最近在Devaraj实验室发现的一种生物中间相系统正在被开发成一种合成细胞室，能够模拟细胞的高度拥挤环境，并实现生物产品的高速率和出口。表面活性剂可以在水介质中形成微米级的海绵中间相液滴。这些结构被称为脂质海绵，以反映其海绵状内部网络和吸收和保留生物分子的能力。由于高的内表面积和多孔纳米结构，脂质海绵可以自发地包封大量的染料和小分子。脂质中间相的多孔连续结构使得能够容易地运输进出液滴，克服了困扰基于脂质囊泡的合成细胞研究的关键问题之一。该项目的具体目标是：（1）通过研究表面活性剂二元混合物形成的纳米结构来设计脂质海绵液滴的物理化学性质，目的是提高稳定性和均匀性，以及（2）实现液滴内生化途径的可编程区室化。目前正在研究两种关键的生化途径：碳固定，以证明在液滴内提供更高水平的CO2并改善反应动力学的能力;以及通过封装DNA可编程TX-TL（转录-翻译）系统来控制蛋白质合成和释放。该项目有可能通过实现一种新型的模块化和可编程的细胞器来显著推进自下而上的合成细胞设计，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评估来支持。影响审查标准。

With this award, the Chemistry of Life Processes Program in the Division of Chemistry, as part of the Rules of Life (RoL): Design and Engineering of Synthetic Cells and Cell Components (DESYN-C3) initiative, is funding Dr. Neal Devaraj from the University of California, San Diego, to investigate lipid "sponges" as programmable compartments for application to the design of synthetic cells. This work capitalizes on an exciting and unexpected finding from the Devaraj lab, that biomimetic materials can be programmed to absorb biomolecules out of solution and concentrate them in a sponge-like interior. Synthetic cells have the promise to revolutionize biomanufacturing by overcoming inherent limitations faced by living cells, for instance their inability to withstand harsh conditions and toxins. Similar to organs in the human body, living cells have evolved to have interior compartments known as organelles, which have specific functions and assist in cell maintenance and function by concentrating reactants or separating mutually incompatible reactions. However it is unclear how much spatial organization and compartmentalization is necessary for the construction of a synthetic cell. Lipid sponges can help answer this question because they can be programmed to trap diverse classes of biomolecules and reactions. This project is training graduate students in supramolecular chemistry, biochemistry, soft matter, and molecular biology. The work is also contributing to outreach activities that are introducing the concept of synthetic cells to the broader San Diego educational community, with the aim of stimulating the entry of low-income and underrepresented student populations into STEM fields. The studies are providing unique insight into how compartmentalization can assist the complex chemical reactions that govern life. Living cells possess an astoundingly high macromolecular concentration. Confinement and crowding effects play critical roles in the kinetics of gene expression, protein folding, and enzymatic reactions. It has been extremely challenging to achieve reproducibly high concentrations of macromolecules inside conventional synthetic cell models such as vesicles. A lipidic mesophase system recently discovered in the Devaraj lab is being developed into a synthetic cell compartment capable of mimicking the highly crowded environment of a cell and achieving high rates and export of biological products. Surfactants can form micron-sized sponge mesophase droplets in aqueous media. These structures are termed lipid sponges to reflect their sponge-like interior network and capacity to absorb and retain biological molecules. Thanks to the high internal surface area and porous nanostructure, lipid sponges can spontaneously encapsulate high quantities of dyes and small molecules. The porous continuous structure of the lipid mesophase enables facile transport into and out of the droplets surmounting one of the key issues that has plagued lipid vesicle-based synthetic cell studies. The specific aims of this project are: (1) engineering the physico-chemical properties of lipid sponge droplets by studying the nanostructures that are formed from binary mixtures of surfactants, with the goal of improving stability and uniformity, and (2) achieving programmable compartmentalization of biochemical pathways within the droplets. Two key biochemical pathways are being studied: carbon fixation, to demonstrate the ability to supply increased levels of CO2 within a droplet and improve reaction kinetics; and controlled protein synthesis and release, through encapsulation of a DNA-programmable TX-TL (transcription-translation) system. The project has the potential to significantly advance bottom-up synthetic cell design by achieving a novel modular and programmable organelle for incorporation into artificial cells, and is providing a valuable model system for the studying the effects of colocalization and sequestration on biochemical reactions.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.