Andrew E. Short 

BioE PhD Defense Presentation 

Date and Time: Thursday, May 4th, 2023, at 1:00 PM 

Location: Krone EBB - Children's Healthcare of Atlanta Seminar Room 

Zoom Link: https://gatech.zoom.us/j/9682106999 

Advisor: 

Corey J. Wilson, PhD (Georgia Institute of Technology) 

Committee: 

Ravi Kane, PhD (Georgia Institute of Technology) 

Manu Platt, PhD (Georgia Institute of Technology) 

Matthew Realff, PhD (Georgia Tech Research Institute) 

Eric Vogel, PhD (Georgia Tech Research Institute) 

Interception of Recombinase Function: Repurposing Transcription Factors as Post-Translational Regulators of Genetic Memory 

Recombinase-based control of gene expression allows for stable genetic memory storage through targeted DNA modifications, facilitating applications in biological information storage, genetic logic circuit design, genome engineering, biomanufacturing, gene and cellular therapeutics, and beyond. This thesis work develops a novel method called "interception" to enhance control over recombinase activity to address some of the drawbacks to recombinase-based memory circuits by reducing metabolic burden, accelerating recombination, and enhancing recombinase compatibility with traditional transcriptional logic circuits. Interception repurposes transcription factors (TFs) to block the post-translational function of recombinase enzymes (instead of TF’s usual role of blocking RNA polymerase-mediated gene transcription), to our knowledge the first such use of TFs. We demonstrate that interception enables inducible control over whether recombination reactions occur.  We explore the use of a set of modular TFs with orthogonal DNA-binding domains and orthogonal inducers for interception control, expanding the number of memory operations controllable via interception. Interception is shown to enable faster genetic memory operations than traditional repression-based control, broadening potential use cases or recombinase-based genetic memory. Lastly, interception is investigated as a method to independently control the recombination reaction of pairs of orthogonal attachment sites with different matched central conserved regions. This approach enables a single recombinase to perform multiple memory operations simultaneously, lowering the metabolic burden of the genetic memory circuit and freeing up cellular resources for additional genetic circuit components. Overall, the work presented here has implications for developing predictable and controllable memory storage in living cells and can be utilized in the design of more complex genetic circuits in synthetic biology.