Synthetic Biology for Knockouts, Knockdowns, and Phospho-Sensing in Diverse Bacteria

Abstract

Biology is defined by experimental stories. These stories, however, are limited by the technologies that are used to tell them. In microbiology, there are limits on the types of organisms we can use, the therapeutic outcomes of anti-microbial treatments, and the enzymatic activities that can be sensed in vivo. My Introduction describes these limitations, and the rest of my dissertation presents my work developing engineering-solutions to them. In Chapter 2, I present research towards a Potentially Organism-Agnostic Knockout (POAK) system. Our ability to knockout genes limits the number of bacteria that are tractable, and I demonstrate that POAK can expand the number of species that we can work with. In Chapter 3, I discuss efforts towards DEcreasing the Selective Pressure Of phage Therapy (DESPOT). That chapter deals with a novel approach towards bacterial infections that aims to have therapeutic benefits without selecting for resistant bacteria. In Chapter 4, I detail a set of potentially Host Organism-Agnostic Kinase Sensors (HOAKS). Current technologies do not allow single cell measurement of serine/threonine kinase activity in bacterial cells. In that chapter, I present two tools that can perform these measurements. These three sets of tools, POAK, DESPOT, and HOAKS, expand the microbes we can work with, the infections we can treat, and the biology we can sense.