Tas Ahmed
BME PhD Defense Presentation
Date: 2023-12-05
Time: 2:00 PM-4:00 PM
Location / Meeting Link: EBB CHOA
Committee Members:
Shuichi Takayama, PhD (Advisor) Blair K. Brettmann, PhD Felipe G. Quiroz, PhD Philip J. Santangelo, PhD Mark P. Styczynski, PhD
Title: Analysis of liquid-liquid phase separated systems for artificial cell applications
Abstract:
Protocells are promising tools for addressing clinical needs at the point of care, particularly when sample is minimal and technological capabilities are limited. Liquid-liquid phase separation of bioinert, low-cost and environmentally friendly polymers have previously been used to form protocells for this application, but underlying study of the specific polymers and the roles in which composition, interaction and enzymatic activity play are limited. In this thesis, we address this gap for two model protocells, a polyethylene glycol (PEG)-polysucrose (Ficoll) system and an adenosine triphosphate (ATP)-polydiallyldimethylammonium (PDDA) coacervate. We first address the shift in thermodynamics of PEG-Ficoll protocells that operate in a specialized cell-like buffer meant to facilitate transcription-translation reactions. This involves creation of a ternary phase diagram of the system through cloud point titration and tie line analysis, and further examination of interaction strengths and solvent quality as probed by fluorescence correlation spectroscopy (FCS) and theoretical analyses. The cell-like buffer lowers the threshold to phase separation, enacting binodal shifts through modulation of excluded volume in Ficoll. Partitioning of nucleic acids, essential to protocell function, are found to change phase preference in response to various protocell conditions, and the results are supported by atomic force microscopy imaging of DNA structural changes. We then shift to ATP:PDDA coacervates in order to study dynamics of a model diffusion-limited enzyme while compartmentalized compared to in bulk. Using FCS, diffusion of the enzyme dextranase is quantitated within coacervate droplets and is studied with respect to droplet viscosity and coacervate composition. Scaling changes in diffusion with respect to polymeric regime are found to be altered through catalysis and substrate inhibition. The work presented in this thesis sheds light on the fundamental variables that affect protocell operation, and the results suggest that engineering of polymer interactions through buffer selection, of protocell composition and of enzyme diffusivity could dramatically affect protocell success.
