Project Summary / Abstract The ability to visualize specific gene expression, exemplified by methods like green fluorescent protein (GFP) reporters, has been pivotal in scientific research over the past few decades. However, most of these techniques rely on optics, and are therefore limited by the ~1 mm penetration depth of light into biological tissues. It is in this context that a class of air-filled protein nanostructures named gas vesicles (GVs) were developed as the first acoustic reporter genes, which enabled the use of ultrasound to image gene expression in cells located in centimeter-deep tissues. Since then, biomolecular engineering of GVs has rapidly expanded the use of these unique air-filled protein nanostructures into directions such as spatially cellular control, drug delivery, pressure sensing, gas delivery, neuromodulation, and multimodal imaging. Despite the rapid progress in the biomedical applications of GVs, the understanding of the genetics and biophysics underlying GV formation lags behind, and it has been recognized that the assembly of GVs in non-native host organisms is far from optimal compared to that in the native host cells. This gap poses a significant, universal obstacle in almost all GV-based technologies, strongly hindering their adaptability and application. Our research program aims to address this challenge by pursuing two parallel directions. In the first, we delve into the fundamental science to unravel the molecular mechanism of GV assembly. In the second, we focus on engineering control, using synthetic biology strategies to design genetic constructs that achieve optimal GV assembly. If successful, the discoveries from these projects will not only enhance our fundamental understanding of this intriguing class of protein organelles, but also revolutionize the biomolecular engineering of GVs, which will generate a major impact for all the biomedical applications of this class of nanostructures in molecular imaging, cellular control, theranostic nano-agents, and biosensing.

Project Narrative Imaging specific biological processes in cells is a fundamental capability for researchers and clinicians in the disease diagnosis and monitoring of therapeutic cells. This proposal builds on the recently introduced ultrasound- based reporter genes, which may enable one to use ultrasound to follow the state of engineered cells in human patients. Specifically, the project aims to develop the next-generation genetic design of these reporters to enable order-of-magnitude increase of the brightness of their ultrasound signals.