Informing Therapeutic Approaches For Infectious Disease And Heart Disease, Focusing On Cell-Free Synthesis Of Bacteriophages And Calcium Transport By Serca

Abstract

My thesis work is divided into two periods: research in the School of Physics and Astronomy, advised by Dr. Vincent Noireaux (Ch. 1-3), followed by research in the Department of Biochemistry, Molecular Biology and Biophysics, advised by Dr. David Thomas (Ch. 4). In Chapter 1, I cover the functional scope and optimizations of the cell-free transcription and translation (TX-TL) platform developed in the Noireaux lab, as described by my 2016 ACS Synthetic Biology publication. This work discusses the breadth of synthetic biological applications available with a cell-free TX-TL system, such as the programming and execution of biological circuits, the bottom-up synthesis of functional biological systems, and the development of a minimal cell system. My work was focused on the cell-free synthesis of infectious bacteriophage particles. I performed parameter space optimization of biochemical reaction components for multiple phage systems. Chapter 2 describes the cell-free synthesis of bacteriophage T4, one of the most complex systems to be fully recapitulated within a cell-free platform, reproducing my 2018 Synthetic Biology publication. In Chapter 3, I share my 2017 peer-reviewed scientific video journal for The Journal of Visual Experiments where I describe the protocol for general cell-free bacteriophage synthesis. Chapter 4 presents my work in the Thomas Lab, where I utilized site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to investigate the structural model by which SERCA, PLB, and DWORF regulate intracellular calcium transport in cardiac muscle. I detail the methodology behind purifying and co-reconstituting these membrane proteins in vesicles, and I present the dynamical information of these proteins obtained by EPR spectroscopy. My experimental background in cell-free synthetic biology, protein dynamics, and magnetic resonance has revealed to me the exciting nature of biophysical investigations. The societal impact and importance of progress in this field has never been more apparent than it is now, in the face of a novel pandemic. After my PhD work at the University of Minnesota, I will continue research in biophysics on the computational side, working as a Data Scientist for the National Institute of Allergy and Infectious Disease, where my new position begins in September.