Title:

Large Scale Enzyme Production for Topologically Controlled dsDNA Hydrogels

Double-stranded DNA (dsDNA) is a basic polymer that is often overlooked in biomaterial synthesis. It comes in a wide variety of sizes while retaining water solubility, making it an attractive material for water-based biomaterials such as hydrogels. Another feature that makes dsDNA an attractive polymer is its ability to be altered enzymatically. This work aims to build a scalable enzyme toolbox for precise and selective control of DNA topology, focusing on restriction enzymes for site-specific cleavage (EcoRI-RTX) and ligases for backbone repair (p50-ligase). The RTX tag appended to EcoRI-RTX allows the protein to be purified via calcium precipitation, making it a relatively simple purification. p50-ligase is a fusion of a T4 DNA ligase and the p50 DNA binding domain, and it is purified via Immobilized Metal Affinity Chromatography (IMAC). Another method of enzyme production explored in this project is Cell-Free Protein Synthesis (CFPS). CFPS uses extracted cell machinery, a DNA template, and exogenous substrate to produce protein without the use of cells, shortening the process by days. Proteins produced can be further purified via chromatography without the need for cell lysis. In addition to commercially available kits, we have investigated the use of E. coli lysates for CFPS. Protocols for protein purifications will continue to be optimized to produce pure and functional enzyme, and the CFPS protocol will be optimized to increase yield.

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