Unifying synthetic small regulatory RNAs

RNA is generally known as a messenger to carry genetic instructions from DNA, however, many small RNAs act as regulators controlling gene expression. Over the past two decades, researchers have engineered such regulatory RNAs and have built simple RNA-based genetic circuits in bacteria, nevertheless, the construction of complex RNA-based genetic circuits remains challenging. This project involves developing generalizable design principles by which simple RNA-based genetic circuits can be combined to generate complex genetic circuits. The utility of these complex circuits is demonstrated by engineering bacteria that produce valuable chemicals that contribute to the bioeconomy. In addition, this project provides unique interdisciplinary training opportunities for students, involving biophysics, biochemistry, molecular biology and engineering. An outreach program provides K-12 teachers with the materials to lead their students through lab activities, broadening student participation in science and engineering. The past two decades have witnessed remarkable progress in understanding complex gene regulatory networks by building simpler genetic circuits from the bottom-up. However, RNA-based genetic circuits have seldom been built by integrating different types of RNA regulators that control multiple steps of gene expression (e.g., both transcription and translation). Consequently, RNA-based circuit complexity is limited. In this project, the investigators first build RNA-based gene expression controllers (GECs) that demonstrate consistently predictable behaviors regardless of connected genetic parts. Mathematical modeling is combined with experimental characterization in order to systematically connect different sensors to RNA regulators. Next, insights into the circuit integration rule are obtained first by quantitatively characterizing both integrated genetic programs and individual GECs then by comparing their behaviors before and after integration. Finally, based on the gathered design rules, dynamic pathway controllers are created to autonomously modulate metabolic pathways, demonstrating enhanced biochemical production. This interdisciplinary project provides the research community with a unified, standardized RNA regulator toolbox for fundamental research and biotechnological applications. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.