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Use of Enzymes for Amplified Signal Generation

  • Author(s): Cisneros, Brandon Thomas
  • Advisor(s): Devaraj, Neal K
  • et al.
Abstract

This dissertation will explore two different methods of generating enzymatically-amplified signals in response to cell surface features. The first method uses a variation on traditional catalyzed reporter deposition employing the enzyme laccase to covalently deposit a fluorescent ferulic acid derivative. This enzyme has distinct properties that make it more suitable for use with catalyzed reporter deposition in living systems. The second portion of this thesis focuses on the development of xanthine methylating enzymes as a reporter protein. It demonstrates how synthetic juxtacrine signaling receptors can be used to induce expression of enzymes capable of methylating a variety of xanthines to produce a detectable, biocompatible small molecule reporter. This approach required heterologous expression of many xanthine methyltransferase enzymes in human cells for the first time so that their properties could be evaluated. In addition to being potentially useful as a reporter protein, the use of xanthine methylating enzymes in mammalian cells has potential connections to synthetic biology and the development of synthetic paracrine signaling pathways using small molecules.

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