A self-actuated cellular protein delivery machinery

Owing to increase the knowledge on biology and available tools for genetic manipulations, biological systems are engineered to perform complex tasks. They can be designed to degrade toxic molecules in environment, produce and deliver complex biological drugs, or process and synthesize valuable materials. Hence, the cellular machines hold great promises to solve world problems such as global warming, world hunger, cancer and so forth. However, most of the complex tasks require protein release to extracellular space in a controlled manner. Development of efficient cellular machines is hampered by lack of convenient strategy for controlled protein release as many of proposed secretion systems are limited in a narrow focused application. In this thesis, we are proposing a novel bifunctional self-exciting protein delivery system for broader applications. The proposed protein delivery machine harbours a genetic circuit that is able to display protein-of-interest on cell surface and to secrete to extracellular space in case of need. To do so, we engineered the autotransporter protein Ag43 to display POI with TEV protease recognition site on the cell surface of Escherichia coli (E. coli). The release of displayed POI was achieved and systematically optimized in vitro via addition of purified TEV protease. To accomplish the self-exciting and controlled release of POI by cells, TEV protease was aimed to be expressed and translocated to extracellular space to cleave the recognition site between POI and Ag43 protein. Four different secretion strategies was employed to secrete TEV protease to extracellular space. While cleavage of POI from cell surface can’t be accomplished through secretion of TEV protease by type I system, YebF fusion, and co-expressing lysis gene, codisplaying TEV protease on the cell surface can release the POI. Our data revealed that release of POI can be tuned with controlling the amount of TEV protease on the cell surface. Considering the simplicity of protein display as well as ability to express Ag43 protein in various organisms, the proposed system can be implemented in more complex genetic circuits and used in diverse applications.