Synthetic organization in vitro and in vivo

Abstract

Organized complexity is a hallmark of biology in general, and eukaryotes in particular. This phenomenon abounds across many size scales ranging from tissues to organelles to protein complexes. Scaffold molecules, which facilitate the assembly of protein complexes, serve as guides for organization. These scaffolds encompass a wide variety of materials, including DNA, RNA, and proteins, and are used for engineering metabolic reactions and signaling pathways. However, the structures which have been produced to date are fairly simple in geometry, and in many cases are not compatible with in vivo assembly. Here, I aim to recapitulate biological organization synthetically using nucleic acids and lipids as scaffolds for proteins used as markers and as reaction catalysts, both in vitro and in vivo. In Chapter 1, I discuss a new method for assaying the assembly of DNA nanostructures using next-generation DNA sequencing. In Chapter 2, I explore a new set of DNA nanostructures capable of self-assembly across a wide range of temperatures and conditions. In Chapter 3, I develop a method called distributed cell division counting (DCDC) for counting bacterial cell divisions that utilizes the segregation of self-assembling fluorescent particles, and apply DCDC to measure the growth rate of a native gut microbe in the mammalian gut. In Chapter 4, I discuss a new set of lipid-based scaffolds that can co-localize enzymes in vivo and apply this technique to enhance indigo biosynthesis. Together, these results indicate that self-assembly can be designed to occur under a wide range of conditions, and demonstrate several practical applications of self-assembled nanostructures.