Development of Screening Platforms for Discovery of Ice Nucleators

• Author / Creator

  Kamijo, Yuki

• In this thesis, I describe the development and demonstration of a screening platform for the discovery of ice-nucleators. Ice nucleating agents (INAs) have been used to control the temperature of ice nucleation within a narrow range at high temperature. They play an important role in atmospheric ice formation and plant pathogenesis. However, the relationship between surface chemical structure and ice nucleation activity remains elusive. In the first chapter of this thesis, I briefly reviewed the current INA study and its experimental methods, and challenges.
  The primary focus within Chapter 2 is the development of Freeze-float selection system that is designed to screen the stronger ice-nucleating substances by buoyancy-based separation. This system employed the density-based separation of the sample encapsulated into droplets, so that allows the identification of the molecules that can cause ice-nucleation at different temperatures and separate from others. The system showed the correlation of the droplet freezing temperature and the INA concentration of the sample droplets.
  To extend the screening system of the freeze-float selection and improve the efficiencies and accuracies, in Chapter 3, I investigated the introduction of automated droplets generation system and uniform controlled-rate cooling system to the freeze-float selection. The new platform successfully improved the capacity of the droplet numbers (from 50 to theoretically 1920 droplets) and sample numbers (from 1 cuvette to 48 vials) that can be tested at once. To confirm the accuracy of the screening system, I investigated the detection threshold of the INA concentration. At the threshold concentration, controlled-rate cooling system showed better differentiation accuracies (p = 4.410-7) compared to the commodity freezer (p = 0.83). The enlarged screening setup can lead to comprehensive research for the ice-binding study.
  Finally, I described the screening of ice-binding glycans by combining ice-affinity purification technique “ice-shell purification” and genetically-encoded glycan library; Liquid glycan array. The glycan library was screened and separated into the ice-included and ice-excluded phase and the ice enriched glycans were identified by the deep-sequencing. Further validation of the potential hit was done by plaque forming assay of the colorimetric phage. This platform will complement with different genetically modified library to identify ice-binding molecules.
  In summary, I tested a methodology to screen the ice-binding glycans that were chemically conjugated to phage. The LiGA array of genetically encoded glycans permitted the screening against immobilized and slowly growing ice surface. We showed the first demonstration of the ice-binding glycan screening result analysis and validation test of the LiGA. However the screening validation results were not reproducible, and hit discovery was inconclusive. Further optimization of the screening platform or library with different glycan modification is needed. Although the result was inconclusive, preliminary screening of ice-binding protein demonstrated the feasibility of affinity-based screening. I believe this platform can be easily adapted for the screening of different modifications, genetically-encoded libraries for further ice-binding molecule discoveries.

• Subjects / Keywords

  • Droplets
  • Ice nucleator
  • Ice nucleation
  • High-throughput
  • Screening

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-jmpr-ha43

• License

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