The field of synthetic biology has leveraged engineering tools such as molecular cloning to create new biological components, networks, and processes. While many of these components and networks have been deployed in the cytosol, there is a shortage of systems that utilize the surface of the cell. In order to address this shortcoming, we have created a synthetic, surface-displayed substrate anchor for bacteria. This approach allows us to engineer surface-based synthetic biological systems as a complement to existing intracellular approaches. We leveraged the tools of synthetic biology to display a catalytically active enzyme that covalently bonds itself to benzylguanine (BG) groups. We created a fusion protein allowing us to place human O6-alkylguanine DNA alkyltransferase (hAGT), also known as SNAP, on the surface of a bacterial cell. Initially, we used this synthetic component as a tool for spatially segregating orthogonal synthetic gene outputs by visualizing an extracellular synthetic green fluorescent reporter, SNAP-Cell® 505-Star, simultaneously with an intracellular red fluorescent protein, mCherry. Moreover, we have shown that our construct enables cells to selectively bond to BG-conjugated magnetic beads. As a result, we have demonstrated that surface displayed SNAP facilitates engineering a direct channel between intracellular gene expression and extracellular material capture. In the near future, we believe this magnetic capture can be expanded as a sortable reporter for synthetic biology as a direct extension of this work. Moreover, our work serves as an enabling technology, paving the way for extracellular synthetic biological systems that may coexist orthogonally to intracellular processes. / Master of Science

The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering. / Ph. D.