ENGINEERING A NON-COMMUTATIVE COMBINATORIAL PROTEIN LOGIC CIRCUIT TO CONTROL CELL ORIENTATION

Restricted (Penn State Only)
- Author:
- Chen, Jiaxing
- Graduate Program:
- Bioinformatics and Genomics (PhD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 19, 2023
- Committee Members:
- Dajiang Liu, Major Field Member
Scott Showalter, Outside Unit Member
Nikolay Dokholyan, Chair & Dissertation Advisor
Keith Cheng, Outside Field Member
David Koslicki, Program Head/Chair - Keywords:
- Protein logic circuit
Synthetic biology
Protein engineering
Cell engineering
Src kinase
Chemogenetic
Optogenetic - Abstract:
- Single protein-based devices that integrate signal sensing with logical operations to generate functional outputs offer exceptional promise for monitoring and modulating biological systems. Engineering such intelligent nanoscale computing agents is a challenge, however, because it requires integration of sensor domains into a functional host protein via intricate allosteric networks within host protein structure. The constructed system must be capable of automatically detecting environmental cues and utilizing sensor information to switch protein conformational dynamics, ultimately producing the desired output functions. Here, we engineer both a rapamycin-sensitive sensor (uniRapR) and a blue light responsive LOV2 domain into human Src kinase to create a protein device that functions as a non-commutative combinatorial logic circuit. In our design, rapamycin activates Src kinase resulting in protein localization to focal adhesions in living cells, whereas blue light exerts the reverse effect that inactivates Src translocation. Focal adhesion maturation induced by Src activation reduces cell migration dynamics and shifts cell orientation to align along collagen nano-lane fibers. Utilizing this protein device, we can reversibly control cell orientation by applying the appropriate input signals, a framework that may be useful in tissue engineering and regenerative medicine. Compared to traditional genetic circuits, our protein-based logic circuit functions as a single unit at the post-translational level, thus reducing response latency, decreasing diffusion-mediated signal losses, reducing metabolic burden (metabolic burden describes how many of a host cell’s resources are required to establish engineered proteins or genetic components), and enhancing circuit reliability. This construct is unprecedented as it demonstrates the logical complexity of a non-commutative (a property in which same inputs with various sequential orders generate different results – for example, subtraction is non-commutative, as 1-2 does not equal to 2-1) combinatorial logic circuit, thereby being a proof-of-principle for future protein-based computers.