Biological template directed nanomaterials have unique properties. Under genetic control, they can specifically interact with other macromolecules or inorganics. Moreover, the biologically based materials are easily assembled into nano-, micro- and macro-scales in a hierarchical manner because of their inherent self-assembly characteristics via molecular recognition. By integrating molecular biology, chemistry and materials science, we can control peptide-material interactions with a genetic approach. This approach provides unprecedented opportunities to design and synthesize novel nanomaterials. Bacterial flagella are composed of thousands of flagellin (FliC) proteins through self-assembly. By means of genetic engineering called peptide display, a foreign peptide can be inserted on FliC and finally displayed on the surface of bacterial flagella. At the same time, it is surface-exposed. The bio-engineered flagella can be used as a display tool for extracellular secretions, live vaccines, protein-ligand interactions, peptide display libraries, and so forth. Recently, flagella-templated synthesis and assembly of inorganic nanomaterials have exhibited a variety of promising applications as nanotubes or nanowires.

Chapter 1 is a literature review that introduces the basis and use of genetically engineered flagella, and the recent progress for flagella based synthesis and assembly of nanomaterials is summarized. Identification of domains that are responsible for the nucleation of hydroxyapatite (HAP) is explored in chapter 2 using flagella surface display technique. A bacterial flagellum has a long threadlike structure that is similar to the shape of type I collagen in bone. Because the nucleation of HAP initiates at the hole zone of type I collagen, N-, C-terminal, part of N-, C-Zone around the hole zone, and a central repetitive (Gly-Pro-Pro)8 (GPP8) were displayed on the surfaces of flagella. Some of the negatively charged non-collagen proteins are also considered to be involved in the nucleation process. Eight glutamic acid residues (E8) from bone sialoprotein (BSP), which are considered to be one of the most important promoters for HAP nucleation, were displayed on the surface of flagella. After nucleation in an HAP supersaturated solution, flagella with E8 and GPP8 sequences were found to be nucleated by nano-leveled HAP crystals. After the E8 flagella were assembled into bundles induced by calcium ions, we found the crystallographic c axes of the HAP nanocrystals were preferentially aligned with the long axes of the flagella. This arrangment is similar to the molecular level of bone. The nucleation of HAP with the spontaneous self-assembly of bioengineered flagella is discussed in chapter 3. Using a biomimetic strategy, at a high concentration, the GPP8 flagella can spontaneously self-assemble into parallel ordered structures that resemble the arrangement of type I collagen fibrils with HAP in bone. Meanwhile, the crystallographic c-axes of HAP are parallel to the long axes of the flagella. The biomineralized flagella can support bone marrow stem cell (BMSC) adhesion and growth. In osteogenic media, the flagella promote differentiation of BMSCs toward osteoblasts. In chapter 4, we introduce the BMSCs differentiation on the integrin-binding motif Arg-Gly-Asp (RGD) and 8 glutamic acids displayed flagella scaffold. Our results indicate that the cells are viable on flagella surfaces and show an enhanced growth rate on RGD peptide enriched flagella surfaces. Immunofluorescence and quantitative real-time PCR (q-PCR) analysis revealed that the up-regulation and early expression of osteogenic specific markers (osteopontin, OPN and osteocalcin, OCN) in cells on bioengineered flagella than on those of wild type and control. We propose the surface chemistry and microenvironment generated by flagella can be recognized by BMSCs and trigger osteogenic signaling pathways.

Phosphorylation is a very important post-translational modification and important for mediating the HAP nucleation in bone tissue. In chapter 5, phosphorylation of serines displayed on flagella surfaces by casein kinase II (CK2) is demonstrated. After phosphorylation, the mineralization of bioengineered flagella was highly increased. Moreover, the poly-glutamic acids displayed on flagella exhibited a higher ability for the nucleation of HAP than did poly-aspartic acids. Because collagen is limited to induce apatite formation from metastable calcium phosphate solutions directly, in chapter 6, a collagen-binding motif (CBM) from OPN is displayed on flagella. The flagella can co-assembled with type I collagen to form hybrid bundles with enhanced HAP nucleation ability.

Flagella were also used as efficient biological templates for synthesis of silica nanotubes (SNTs) in aqueous solution under mild conditions. In chapter 7, the morphology of SNTs was tuned by adjusting the pH of the solution. The morphology and surface features of SNTs could also be controlled by the modification of the surface chemistry of flagella with the surface display technique. Finally, a variety of quite different morphologies and surface features of SNTs were obtained. In chapter 8, bilayer TiO2/SiO2 nanotubes mediated by flagella as templates are demonstrated using a new sol-gel method. The reaction was carried out in aqueous solution under ambient conditions. The thickness of either layer is controllable by varying the concentrations of the precursor solution or reaction time. After calcination at 500 °C, the organic flagella templates were removed. At the same time, the inner TiO2 layer became crystalline phases and dispersed inside the SiO2 matrix close to the central pore. The outer SiO2 shell was still amorphous but supported TiO2 nanoparticles as a "skeleton".