Engineering DNA Gels For Cell-Free Protein Production

Abstract

One of the fundamental goals of biological engineering is the harnessing of biological systems to produce desired products. Protein production of exogenous proteins in living cells has long been a staple of molecular biology. However, living biological systems present fundamental limitations as the scientists' desire to produce more complex and varying molecules in cells competes with normal cell processes. Ideally, one can isolate the required cell pathway away from the living system in order to explore the full range of possible molecular permutations allowed by chemistry without the limitations of biology. In vitro protein production allows life's central dogma to be performed outside the confines of a cell, creating the possibility of producing toxic proteins and testing full mutation spaces in the DNA-RNA-Protein pathway. This possibility also opens up the need for materials to interface with genes and protein in an in vitro platform. Our lab has engineered a DNA gel that interacts with the gene-of-interest to increase protein yields while protecting the gene from degradation. DNA has long been investigated as a genetic material, but only in the pat decade has its vast potential as a generic polymer been elucidated. Beyond its monodispersity, the specificity of binding interactions in Watson-Crick base-pairing allows a unique level of control over structure and material organization at the nanoscale. In creating networked DNA structures that can incorporate genes into the gel network, we create protein-producing gels. However, further engineering of DNA gels is required in order to produce a system more comparable to the morphology and functionality of a cell, notably the isolation of gene sets and the ability to connect genotype and phenotype when testing protein activity. This thesis work discusses methods to create cell-size DNA gels that possess the ability to both produce and capture protein, practical considerations for DNA manipulation in cell-free protein production, and provides insight into further functionalization of DNA gels to provide more diverse applications in the context of protein engineering.