Valerie Kay

BioE Ph.D. Defense Presentation

February 5, 2025

1:00 PM

Location: Petit IBB, Suddath Seminar Room 1128

Zoom: https://gatech.zoom.us/j/94255404381

Advisor: Corey Wilson, Ph.D. (ChBE, Georgia Institute of Technology)

Committee:

Dr. John Blazeck, Ph.D. (ChBE, Georgia Institute of Technology)

Dr. Julie Champion, Ph.D. (ChBE, Georgia Institute of Technology)

Dr. Yury Chernoff, Ph.D. (Biological Sciences, Georgia Institute of Technology)

Dr. Brian Hammer, Ph.D. (Biological Sciences, Georgia Institute of Technology)

Mapping Alternate Allosteric Communication of LacI Anti-repressors and Their Application in Saccharomyces cerevisiae

Regulatory proteins are powerful tools that control all life processes. Many regulatory proteins function by allosteric communication, a mechanism in which a signal is propagated between two functional surfaces across the protein. The lactose repressor (LacI) has been essential in unveiling the mechanism behind allostery. It has also been engineered to build anti-repressors of the inverse phenotype (anti-lacs), which together can build Transcriptional Programming (T-Pro) genetic circuits in E. coli. While LacI has been studied in great detail, I posited that the allosteric pathways of engineered anti-lacs differ from their native repressor. To test this hypothesis, I performed deep mutational scanning on two engineered anti-lacs to generate a single-mutation phenotype-genotype heat map for each anti-lac. Through this work, I compared the allosteric pathways of anti-lac transcription factors (TFs) and developed comprehensive design rules for engineering alternate allosteric communication. While these TFs were analyzed in E. coli, I posited that these design rules could be translated to any chassis. I successfully used the design rules developed from this work to build the first engineered anti-repressor in Saccharomyces cerevisiae, a model higher-order eukaryotic organism. Additionally, I developed a T-Pro reporter system in S. cerevisiae by engineering a high-performance promoter capable of regulating transcription with LacI TFs. Finally, by engineering alternate DNA recognition on my engineered anti-lac and on yeast-enhance LacI, I developed the building blocks for T-Pro in S. cerevisiae. This work verifies the importance of understanding the allosteric pathway of LacI TFs and lays the foundation for the design and implementation of more complex T-Pro circuits in S. cerevisiae.