Project Summary / Abstract The ability to image specific gene expression and cellular signaling pathways has long been a powerful tool for scientists, and the methods to achieve this goal, such as the fluorescent reporter genes, are widely used nowadays across different fields of biomedicine. However, the penetration depth of light has limited the use of fluorescent reporter genes in animal models and cell-based therapies in humans. To this end. a group of gas- filled protein nanostructures named gas vesicles (GVs) were recently introduced as the acoustic reporter genes and enabled, for the first time, the use of ultrasound imaging to visualize specific gene expression in cells located at centimeter-deep tissue. GVs are sub-micron particles originally evolved in photosynthetic microbes to achieve cellular flotation, and expectedly, these wildtype GVs do not possess the correct properties that can maximize their detection by ultrasound. This limitation on imaging sensitivity is perhaps the biggest hurdle currently facing the application of the first-generation acoustic reporter genes. In this proposal, we aim to introduce the second- generation acoustic reporter genes by innovating the mechanical properties of the protein shell. In Aim 1, we will leverage the Genetic Code Expansion technology to achieve site-specific crosslinking among the monomeric shell proteins. This is to recognize that the inward buckling of the protein shell holds the key to the sensitive detection of these acoustic protein nanostructures but often leads to the rupture of the wildtype GV shell, and a systematic crosslinking will substantially increase the tensile strength of these protein shells. Specifically, we will systematically search sites that allow the incorporation of non-canonical amino acid and lysine for proximity- induced chemistry, and this search will be performed under the rational guidance of the structural models. In parallel to Aim 1, we will develop finite-element modeling and acoustic measurement method in Aim 2 to understand the effect of crosslinking on the mechanical properties of the shell, which will establish benchmark values and aims for the design of the protein nanostructures. In Aim 3, we seek to establish sensitivity enhancement of these rupture-resistant acoustic protein nanostructures in a rodent model. Our interdisciplinary approach merges synthetic biology, chemical biology, acoustics, and solid mechanics, and if successful, this high-risk high-gain project will lead to a new generation of ultrasensitive acoustic reporter genes that broaden the technology to many therapeutic and diagnostic applications, especially in the functional tracking of cell-based therapies and targeted imaging of biomarkers.