The goal of this research is to develop platforms for the design and construction of tailor-made molecular sensors that will be used for programming dynamic cellular behavior. In particular, these sensors will be used to construct signal integration schemes for programming logical cellular response. In addition, this research includes an integrative educational and research plan that will build new strategies for educating scientists and engineers and lay the foundation necessary for them to push the boundaries of scientific development and technology forward. Specifically, this plan proposes to: (1) develop a high-throughput strategy for the generation of trans-acting, ligand-responsive RNA switches, (2) develop platforms for the design of cis-acting, ligand-responsive RNA switches, (3) integrate these sensor elements into the design of logical processing schemes that program different cellular functions depending on combinations of input signals received, and (4) solidify a strong base in biomolecular and cellular engineering and encourage its advancement by training and educating scientists through cutting-edge, integrated research and educational plans.
Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in industrially-relevant eukaryotic organisms, such as the budding yeast Saccharomyces cerevisiae. We have developed a novel class of ribonucleic aicd (RNA)-based controllers that provide predictable tuning of protein expression levels in yeast. Two libraries of synthetic control modules were generated through cell-based screening strategies, where these modules can be combined to build RNA hairpin elements with rationally programmed gene regulatory activities (i.e., controllers). Structural engineering methods were applied to enhance the insulation and modularity of the resulting components to support their broader use as tools across different genetic circuits. To demonstrate the utility of the RNA controllers, we applied these genetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, which plays an important role in many industrial biosynthesis processes. The studies provided insight into natural control strategies and highlighted the utility of these control module libraries for manipulating and probing biological systems. We subsequently extended this toolset to building a new class of RNA sensing-actuation devices (i.e., RNA switches) based on coupling the synthetic control modules with RNA sensors that bind to specific molecular inputs. We demonstrated a modular assembly strategy for building RNA molecules that can detect specific molecules in the cellular environment and then link these detection events to specific cellular behaviors. We also developed three strategies for tuning the quantitative performance of these RNA switches to optimize their activity for specific applications. The modularity and tunability of this switch platform will allow for rapid optimization and tailoring of this gene control device, providing a useful tool for the design of complex genetic networks in yeast. New curriculum and outreach programs have been developed under this award. Three new courses, targeted to different levels of students, were developed that span engineering, biology, and chemistry. Specifically, an advanced undergraduate biomolecular and cellular engineering laboratory was developed that emphasized open-ended design-based projects. A freshman-level introduction to bioengineering course was developed that introduced students to foundational concepts in biological engineering, including problem solving, analysis, and design, within the context of engineering biological systems. And an advanced undergraduate / graduate-level course in synthetic biology was developed that emphasized advanced concepts and techniques for the design and implementation of engineered genetic systems. In addition, two outreach programs were developed that focus on K-12 education, and specifically bringing cutting edge science and engineering areas into the K-12 classroom and bringing high school students into research laboratories for exposure and training.
