Rapid prototyping and design of cybergenetic single-cell controllers

The design and implementation of synthetic circuits that operate robustly in the cellular
context is fundamental for the advancement of synthetic biology. However, their practical
implementation presents challenges due to low predictability of synthetic circuit design and
time-intensive troubleshooting. Here, we present the Cyberloop, a testing framework to
accelerate the design process and implementation of biomolecular controllers. Cellular
fluorescence measurements are sent in real-time to a computer simulating candidate sto-
chastic controllers, which in turn compute the control inputs and feed them back to the
controlled cells via light stimulation. Applying this framework to yeast cells engineered with
optogenetic tools, we examine and characterize different biomolecular controllers, test the
impact of non-ideal circuit behaviors such as dilution on their operation, and qualitatively
demonstrate improvements in controller function with certain network modifications. From
this analysis, we derive conditions for desirable biomolecular controller performance, thereby
avoiding pitfalls during its biological implementation.