Directed Evolution Approaches for Improved Genetically Encoded Fluorescent Calcium Ion and Voltage Indicators

  • Author / Creator
    Zhao, Yongxin
  • Fluorescent proteins (FP) have revolutionized our imaging technologies and facilitated visualization of biochemical and physiological processes occurring in complex biological systems, which has opened up a new and unprecedented era in cell biology. Although FPs mainly serve as passive fluorescent labels for reporting gene expression and protein localization, FP-based indicators also serve as indispensable tools for dynamic imaging of cellular signaling including neuronal activities. However, few FP-based indicators provide robust performance for in vivo imaging, and the development of reliable FP-based indicators remains a challenging engineering problem, mainly due to lack of structural information for rational design and effective methodologies of protein engineering. The goal of this thesis work is to tackle the long-standing challenge of engineering FP-based indicators for improved performance. This thesis describes a variety of directed evolution strategies to develop FP-based indicators for neuronal activities. First, I developed a colony-based directed evolution method to screen for improved single FP-based calcium ion (Ca2+) indicators. This novel strategy accelerated the engineering of single FP-based Ca2+ indicator and led to several variants with improved performance and various new colors. This palette of new Ca2+ indicators enables simultaneous monitoring of Ca2+ transients in different cellular compartments or different types of cells, which opens up a new era of colorful Ca2+ imaging. Next, by combining microfluidic technology and colony-based screening, I designed an automated cell sorting approach that enables screening variants of FP-based Ca2+ indicator with throughput up to 300 cells/s. This new approach saved considerable time and effort for evolving a new yellow FP-based Ca2+ indicator, Y-GECO. The end product, Y-GECO1, is a useful tool for Ca2+ imaging in cell cultures and brain slices. Finally, I designed a hierarchical screening method to engineer Archaerhodopsin-based voltage indicators with a focus on improving fluorescent brightness. The latest generation of variants, designated QuasAr1 and QuasAr2, shows superior performance and brightness compared to their predecessors. Together with our collaborators, we demonstrated that QuasAr1 and QuasAr2 enable fully optical electrophysiological interrogation of neuronal circuits in intact brain tissues.

  • Subjects / Keywords
  • Graduation date
    Fall 2014
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.