Synthetic biology applications of engineered riboregulation

Abstract

For synthetic biology to make a lasting impact on real-world problems, further increases in the complexity of biomolecular devices are required. Currently, there is a shortage of orthogonal parts that can be assembled to construct highly complex circuits and networks. RNA molecules are a popular source for synthetic biology parts, due to the versatility and predictability of RNA structures. Previously, our lab developed the engineered riboregulator, a RNA-based gene expression system. The advantages of synthetic riboregulation include: physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and the ability to independently riboregulate multiple genes simultaneously using orthogonal riboregulator variants. We performed two sets of in vivo experiments that illustrate these unique features and developed two, higher order synthetic devices based on orthogonal riboregulation: the programmable kill switch and the genetic switchboard. The in vivo experiments involved tracking the localization of the TonB protein and manipulating the SOS DNA damage repair network. These studies highlight the ability of our riboregulator to reveal new insights into microbial physiology. Addressing mounting biosecurity concerns, the programmable kill switch employs two riboregulator variants, which regulate two lambda phage proteins, to induce cell lysis rapidly and selectively. Only when we co-expressed the phage proteins did cell suicide occur, and the circuit can link cell death to four different biological signals. To construct a genetic switchboard, we further increased the number of riboregulators in use by designing two new variants. We directly tested our switchboard in a biosensing setup that reports on four environmental signals in single cells using four differentiable reporters. Finally, we utilized the genetic switchboard in a proof-of-concept metabolic engineering application. The metabolism switchboard regulates four metabolic enzymes that control carbon flux through three, E. coli glucose utilization pathways, and we measured its impressive performance across the RNA, protein, and metabolome scales. All together, the applications described here showcase the considerable real-world potential of the engineered riboregulator.