✍🏼 Brianna Jayanthi, Bhagyashree Bachhav, Zengyi Wan, Santiago Martinez Legaspi, Laura Segatori

🏠 Rice University, Houston, TX

📑 Synthetic Biology (2021)

Abstract
Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

How is the WOLF used in this study
The authors used a WOLF cell sorter to generate stable HEK293 cell lines expressing different levels of GFP prior to their experimental studies. After transducing HEK293 cells with a GFP-expressing lentiviral vector, they analyzed the resulting heterogeneous population on the WOLF sorter and sorted cells based on distinct levels of GFP fluorescence to isolate individual cells with low, intermediate, or high GFP expression. These sorted cells were then seeded as single cells into 96-well plates, expanded into monoclonal lines, and characterized by flow cytometry to select three stable GFP-expressing cell lines. The purpose of using the WOLF sorter was to establish well-defined GFP expression cohorts that served as foundational reporter cell lines for subsequent experiments investigating nanobody-mediated protein localization and control of subcellular targeting.