Characterizing and controlling biological processes in single cells and in vitro by integrated perturbation approaches.

摘要

This thesis devoted to the development of a general chemical perturbation method, a spectroscopy that allows quantitative characterization and control of biological function. This perturbation and response approach involves driving nonequilibrium biological systems via temporal excitation with pulsatile chemical waveforms. Applications to a range of physiological processes are demonstrated, including cell reproduction, cell-surface interaction, macromolecular organization, and facilitated protein translocation. The first two chapters (Chapters 2 and 3) present an integrated experimental and simulation study on the response of Caulobacter crescentus cell cycle regulatory network to perturbation of a histidine kinase, DivJ, and the correlation of this perturbation to various phenotypic outputs including cell cycle timing and noise, cell-surface adhesion, and motility. This study demonstrates the use of chemical perturbation for better understanding the function of network components within a complex regulatory network. In Chapters 4 and 5, we developed a multiple-pulse chemical perturbation spectroscopic approach to characterize and drive the cell cycle oscillator of Caulobacter crescentus . We measured the phase response curves of the cell cycle oscillator that is subjected to perturbation of a key response regulator protein CtrA and unambiguously demonstrated the connectivity between phase response and phase locking (i.e. synchronization) for the cell cycle oscillator in Caulobacter crescentus through the integration of experiment and theory. In the final chapter, the focus shifts from in vivo studies to an in vitro system, where we utilized the flow perturbation to investigate the target searching mechanism of a DNA repair protein on duplex DNA. In addition to the characterization of single protein thermal diffusion dynamics under very dilute single-molecule assay conditions (i.e. one protein at a time), we developed a "speckle" assay to study the protein-DNA interaction under near physiologically relevant conditions. This study allowed addressing questions of crowding and the effect of protein-protein interactions.