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

作为生命规则(ROL)：合成细胞和细胞组件的设计和工程(DESIN-C3)倡议的一部分，化学系生命过程化学项目将资助加州大学圣地亚哥分校的Neal Devaraj博士将脂肪“海绵”作为可编程隔室进行研究，将其应用于合成细胞的设计。这项工作利用了Devaraj实验室一项令人兴奋和意想不到的发现，即仿生材料可以通过编程从溶液中吸收生物分子，并将它们浓缩在海绵状的内部。合成细胞有望通过克服活细胞面临的固有限制，例如它们无法承受恶劣条件和毒素，来彻底改变生物制造。与人体的器官类似，活细胞已经进化成具有被称为细胞器的内部隔间，这些细胞器具有特定的功能，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，目前尚不清楚建造合成细胞需要多少空间组织和分区。脂肪海绵可以帮助回答这个问题，因为它们可以被编程为捕获不同类别的生物分子和反应。该项目正在培养超分子化学、生物化学、软物质和分子生物学方面的研究生。这项工作还有助于向更广泛的圣地亚哥教育界介绍合成细胞概念的外联活动，目的是鼓励低收入和代表性不足的学生进入STEM领域。这些研究为划分如何有助于控制生命的复杂化学反应提供了独特的见解。活细胞拥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)中实现可重复的高浓度大分子一直是极具挑战性的。Devaraj实验室最近发现的一种脂类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，实现生物制品的高产量和出口。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，以反映其海绵状的内部网络以及吸收和保留生物分子的能力。由于高内表面积和多孔性的纳米结构，脂肪海绵可以自发地包裹大量的染料和小分子。脂质中间相的多孔连续结构使其能够方便地进出液滴，克服了困扰基于脂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类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)实现液滴内生化路径的可编程划分。目前正在研究两条关键的生化途径：碳固定，以展示在液滴中提供更多二氧化碳的能力，并改善反应动力学；以及通过封装DNA可编程的TX-TL(转录-翻译)系统来控制蛋白质的合成和释放。该项目有可能显著推进自下而上的合成细胞设计，实现了一种新的模块化和可编程细胞器，用于整合到人造细胞中，并为研究共址和隔离对生化反应的影响提供了一个有价值的模型系统。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.