The ability to efficiently sense and respond to molecular signals is crucial for any organism's survival. Sensitive, specific and timely, microbial sensing systems provide an excellent starting point for the engineering of single-celled organisms to detect new molecules of human interest. Towards this goal, our lab pursues the engineering of diagnostic organisms - single celled sensors that detect and respond to human biomarkers just as any medical diagnostic would. Specifically, my work focuses on the development of a yeast-based biosensor as a diagnostic for chronic kidney disease.Baker's yeast, Saccharomyces cerevisiae, has a well-studied detection system to sense and respond to environmental cues. The crux of this detection system is Ste2p, a G-protein coupled receptor (GPCR) that detects α-factor, an extracellular peptide hormone, and ", 'consequently initiates an internal response in the yeast cell. This internal response can be converted to an output detectable by a human user. My work contributes to knowledge gaps in the development of yeast-based biosensors as medical diagnostics by demonstrating a strategic approach to engineering a yeast membrane receptor to detect an orthogonal peptide biomarker.', 'To address these knowledge gaps, my work is divided into three main studies. The first study establishes a high-throughput directed evolution protocol to evolve yeast membrane receptors with non-native detection capabilities. As a proof of concept, this directed evolution protocol is used to evolve Ste2p to detect peptides that differ by one amino acid as compared to the α -factor peptide.', 'The second study builds upon the developed evolution method and describes efforts towards engineering a yeast based biosensor for a peptide biomarker of chronic kidney disease. A yeast receptor was evolved to detected a C-terminally amidated version of this biomarker. The evolution of the receptor was made possible through a step-wise directed evolution approach. The yeast biosensor detected the amidated biomarker in pooled human urine, highlighting the potential of yeast-based biosensors to be used in clinical settings. This study also interrogates the contributions of mutations accumulated over the course of directed evolution towards overall receptor performance and examines genetic changes to enhance the diagnostic performance of the strain.', 'Lastly, the third study explores the use of deep scanning mutagenesis on Ste2p. The evolved receptors from the second study exhibited hyperactive activity, and deep mutational scanning was performed on Ste2p to identify single amino acid residues in Ste2p that contribute to hyperactive activity. Though the scan did not reveal novel single mutations that contribute to hyperactivity, the study describes the optimization of a one-pot protocol to develop a library of single amino acid mutants, possible reasons as to why the scan for hyperactivity did not yield previously unknown mutations and alternative future experiments where a deep mutational scan of Ste2p could find use in the engineering of Ste2p to detect non-native peptides.

Last modified

• 09/30/2019

Creator

• Adebola Adeniran

DOI

• https://doi.org/10.21985/n2-0abc-5m65

Subject

• Chemical and Biological Engineering

Keyword

• Bioengineering

Date created

• 1/1/2018

Resource type

• Dissertation

Rights statement

• In Copyright