Abstract
Mammalian synthetic biology aims to rationally program the behavior of cells with synthetic molecular
circuits, and it holds great promises for diverse biomedical fields such as cell fate reprogramming and oncolytic
virology. Synthetic circuits have been predominantly constructed with transcription factors, and delivered on
DNA-based vectors that are compatible with transcriptional regulation but may insert into and mutagenize the
host genome. Protein-level circuits would potentially operate faster, compute in parallel in subcellular
compartments, and interface directly with cell endogenous inputs/outputs. They would also enable the
development of RNA-based vectors with lower mutagenic risks, because protein-level circuits can serve as
both cargos that functions properly even when expressed from an RNA vector, and as controllers for RNA
viruses through regulation of essential viral proteins. However, despite researchers' efforts, protein circuits
have been limited to a few ad hoc examples, because existing protein components, unlike transcriptional units,
lack composability (the ability to select and assemble modular building blocks differently for different tasks).
In our preliminary study, we successfully engineered viral proteases as composable elements for
protein-level circuits, and demonstrated a broad variety of functions. Building upon the initial success, I will
enhance the capability of protease circuits, and concurrently develop a RNA vector for their delivery. To better
couple protease circuits to cell endogenous inputs/outputs, I will build detection modules that converts specific
proteins' presence and signaling events into protease activity, and execution modules that knock down
endogenous proteins. To meet a larger input/output palette with expanded computing capacity, I will mine more
orthogonal proteases and engineer them to be regulatable by other proteases. Meanwhile, to facilitate the
optimization of more complex circuits and to explore its unique features compared to traditional transcriptional
circuits, I will establish a computational framework for simulating protease circuits. As for delivery, I will
engineer a negative-strand RNA virus into a vector by regulating essential viral proteins with protease circuits
to achieve safety and cell-type specificity. I will also test the limit of the vector's cargo capacity and explore
strategies to raise the limit. All told, my project will engender a more powerful platform for constructing and
delivering DNA-free synthetic circuits into mammalian cells.
My career goal is to lead my independent research group devoted to establishing a general-purpose
toolkit for non-mutagenic manipulation of mammalian cells. During the K99 phase, I will continue to receive in-
depth quantitative and mathematical training from Dr. Elowitz and our collaborators, and acquire key
experimental techniques from my consultants. I will also have demonstrated the feasibility of the basic designs
behind each aim. My trained skills, as well as individual designs, will come together in the R00 phase, give rise
to more complex circuits with direct biomedical relevance, and lay the foundation for my future career.