Browsing by Author "Bennett, George N."

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Controlling bioenergetic systems using protein design and synthetic biology.
    (2019-03-27) Atkinson, Joshua T; Silberg, Jonathan J.; Bennett, George N.
    An understanding of the mechanisms that life uses to regulate this flow of energy and how to program them at different scales is becoming of great importance for the field of synthetic biology as researches build living systems with ever increasing complexity. My thesis goals are to determine design rules for programming the function of proteins that control energy charge and electron transfer in cells. Herein, I describe my efforts in developing a computational pipeline for analyzing sequencing data from bacterial growth selections that depend on the function of adenylate kinase, a protein that controls cellular energy charge, applying this pipeline to libraries of topological mutants to uncover trends in how the energetic frustration in an allosteric domain relates to tolerance to increased local conformational entropy, developing a high-throughput growth selection for monitoring the efficiency of an electron transfer pathway in vivo, design of synthetic allosteric metalloprotein switches to control electron transfer in the cytosol of cells, and finally coupling cytosolic metabolism to a synthetic extracellular respiratory circuit that enables the transfer of intracellular electrons to surface of cells for reduction of conductive materials. These studies help enable synthetic biology strategies for the control of bioenergetics across a variety of length scales including from local energetics of protein structures to the energy charge of the cell to the energetic interface of cells and materials.
  • Thumbnail Image
    Item
    Development and Characterization of Split Methyl Halide Transferase for Biosensor Applications in Soil
    (2019-09-10) Huh, Dongkuk; Silberg, Jonathan J.; Bennett, George N.
    Soil microorganisms contribute to agricultural productivity by forming symbiosis with plant roots and affecting the solubility of soil nutrients. Microbiological activity in soil can be monitored directly by coupling the expression of a methyl halide transferase (MHT) to a conditional reporter. This unique bioreporter can provide information about microbial sensing and behaviors without disrupting soil. However, MHT reporting has only been used to report on slow transcriptional processes, limiting its utility. To overcome this challenge, I have created a split MHT (SMHT) that consists of two MHT fragments that are only functional when reconstituted. This SMHT was rationally designed using family sequence information and shown to exhibit MHT-fragment complementation upon fusion to different pairs of interacting proteins. Furthermore, a rapamycin biosensor was constructed that links the detection of rapamycin, a secondary metabolite synthesized by a soil microorganism, to SMHT fragment complementation. The SMHT should be generally useful for reporting on conditional protein-protein interactions in hard-to-image soils and sediments.