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

University Of California-San Diego, La Jolla CA

Investigators

Abstract

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.