Abstract
Synthetic biology offers great promise to a variety of applications through the forward
engineering of biological function. Most efforts in this field have focused on employing living
cells. Cell-free approaches, on the other hand, offer simpler and more flexible contexts, but few
synthetic systems based on cell-free protein expression have been constructed. Here, we
evaluate cell-free regulatory systems based on T7 promoter driven expression, and we
demonstrate negative feedback, an essential motif in many natural and engineered systems.
First, we characterize variants of TetR and LacI repressible T7 promoters in a cell-free context
and examine sequence elements that determine expression efficiency. Then, we explore different
approaches for composing regulatory systems, leading to the implementation of inducible
negative feedback in E. coli extracts and in the minimal PURE system, which consists of purified
proteins necessary for transcription and translation. Our quantitative cell-free component
characterizations and demonstration of negative feedback embody important steps on the path to
harnessing biological function in a bottom up fashion.
