Tools4Cells: Development of self-amplifying RNA systems for bacterial protein synthesis

An award is made to William Marsh Rice University to enable the development of a safe, programmable RNA-based system for transient protein production in bacteria. Harnessing the replicative machinery of single-stranded RNA bacteriophages and incorporating regulatory control elements, this project will produce self-replicating RNA (srRNA) scaffolds that function across a broad range of bacterial species. This platform enables precise, short-term protein production without the risks of horizontal gene transfer or permanent genomic integration. The technology has wide-ranging applications in environmental remediation, sustainable agriculture, and therapeutic delivery by enabling temporary, task-specific microbial functions with improved biosafety. Through partnerships with academic institutions, industry, and non-profit organizations, the project will contribute to global efforts in environmental sustainability, food security, and public health. The resulting srRNA toolkit will be openly shared with the scientific community to promote innovation, cross-sector collaboration, and responsible biotechnology development. The intellectual merit of the research lies in its transformative approach to overcoming key limitations of existing RNA-based expression systems in bacteria. Current srRNA platforms are largely restricted to E. coli, limiting their broader utility. This project will engineer a diverse set of srRNA scaffolds derived from newly characterized RNA bacteriophages to extend compatibility across a wider range of bacterial hosts. It also addresses longstanding technical challenges such as cell toxicity due to uncontrolled RNA replication by integrating regulatory elements, such as riboswitches, to provide precise temporal control over RNA replication and protein synthesis. By bypassing the need for DNA and transcription, the system enables rapid, direct protein production and ensures complete degradation of constructs after use. Such features are especially valuable in field-based or ecologically sensitive applications. Together, these innovations unlock new capabilities for safe and targeted microbiome manipulation and facilitate the development of novel applications in microbial and environmental biotechnology. 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.