Ph.D Thesis


Ph.D StudentAdir Omer
SubjectEngineering of Light-Based Communication Pathways in
Synthetic Cells
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor ASSOCIATE PROF. Avi Schroeder


Abstract


The field of synthetic cells (SCs), micron-sized constructs designed from the bottom-up, is rapidly developing with potential applications for basic and translational research. Recently, SCs have been used as models to investigate the origin of life, isolate and study cellular processes and as potential therapeutic and diagnostic systems. Controlling cellular processes inside SCs and integrating them with living tissues is important for realizing these applications, particularly for the utilization of SCs as drug-producing micro-factories inside the body. Engineered chemical communication pathways have been applied in SCs for intracellular and intercellular signaling, enabling cross talk between SCs and natural cells, as well as signaling within SC populations. Nevertheless, the implementation of increasingly complex functions in SC technologies requires multiple orthogonal communication pathways that provide multichannel signaling opportunities. Light for example, offers an attractive way for regulating cell functions, but its use is limited by its restricted penetration depth into tissues.

In this thesis, a novel signaling method based on bioluminescence was engineered in SCs to self-activate cellular processes and establish communication with natural cells. First, the SCs' membrane and internal compositions were optimized to maximize light emission through the production of Gaussia luciferase. Correct folding of luciferase proteins in SCs was achieved after adjusting their redox potential to support the formation of the proteins' internal disulfide bonds. Next, the engineered SCs demonstrated their ability to communicate with innate fungal cells through bioluminescence and activate fungal sporulation in a quorum-sensing like mechanism. To achieve intracellular signaling in SCs, self-activating fusion proteins were designed, employing bioluminescence resonance energy transfer to activate an inherent light-responsive domain using the bioluminescent oxidation reaction of luciferase. These proteins were integrated into SCs and mediated bioluminescent controlled transcription and membrane recruitment of proteins.

The signaling functionalities developed in this thesis lay the groundwork for utilizing SCs as embeddable light sources that stimulate cellular processes inside tissues with high temporal and spatial resolution, setting the stage for advancing from single SCs to synthetic tissues and controlling future therapeutic and diagnostic capabilities in SCs.