INTELLECTUAL MERIT: This proposal aims to develop the filamentous bacterial virus M13 as a component for the assembly of higher-order structures. To accomplish this the PI proposes the following specific tasks: (1) Systematically explore relationships between fluorochrome density, brightness, and quenching for single-color fluorochromes linked to the phage protein coat in order to optimize the ability to image and track individual phage. (2) Use cross-linking chemistries to link FRET (Forster Resonance Energy Transfer) dye pairs and other functional groups to phage and study energy absorption and transfer along the extended protein-DNA backbone in order to lay the groundwork for applications involving photonic energy absorption and transfer. (3) Develop a collection of phage that bind specifically to diverse substrates such as nickel, plastics, silicon, or semiconductor materials in order to target phage SAMs (self assembled monolayers) to unique surfaces. (4) Develop ways to join phage using biomolecular affinity interactions, such as biotin-streptavidin binding or peptide coiled-coil formation, to link phage to each other and around hubs in defined geometries in order to build on these SAM ?foundations?. (5) Study the structural bases of wool-like and cable-like phage networks using fluorescence, atomic force, and electron microscopies. (6) Extend these aggregate measurements to the measurement of individual phage spectral intensities using digital fluorescence microscopy to validate the use of individual pH-sensitive-dye-labeled phase particles (pHages) as tools for the measurement of pH values on ultrafine spatial scales and incorporate these sensors into our higher order assemblies.
BROADER IMPACTS: This RUI project will take place at Haverford College, a small liberal arts institution with a diverse student body and a strong tradition of undergraduate student involvement in research. The liberal arts environment is a rich training ground for the nation?s future leaders in science and medicine, and the opportunities made possible by this project will expose students at all levels to the research process. These effects will be magnified by outreach activities involving secondary school students and their teachers through both off-campus talks and on-campus workshops, and will enhance the research-rich environment at the College through support of this interdisciplinary project. Between 4-6 students per year will be involved in accomplishing the objectives of this proposal. The research team will include both underclass students and seniors in the process of doing science over the summer and during academic terms. The training aspects of this proposal develop the human component of US scientific infrastructure by reaching students at a critical point in their careers where they are learning new things and making decisions about future directions. These students will be full participants in this project by their involvement in these experiments, their presentation of their work at national meetings, and their co-authorship on publications in leading journals describing their contributions to this shared endeavor. In a previous RUI grant to the PI involving a total of 30 students, 70% of the undergraduate trainees were female and 43% were members of underrepresented groups. The current project expects to continue these efforts to broaden participation in the nation's scientific enterprise.
Biological systems offer rich sources of inspiration for the development of novel self-assembling architectures for applications in materials science. Whether the contractile array of a muscle cell, the mitotic apparatus of a stem cell, or the flagella of a swimming green alga, living systems abound with examples of highly intricate structures built "from the bottom-up" via specific protein-protein interactions. Although each individual protein piece may be only nanometers in dimension, together they assemble into architectures that can span multiple length scales, including objects visible with the unaided eye. In this project, we investigated using bacterial viruses (bacteriophage, phage) as prefabricated bio-components for the construction of larger, ordered arrays. A bacteriophage can be viewed as a self-replicating DNA instruction set that can infect a cellular "operating system" to produce many progeny virus, each a copy of that instruction set packaged in a sheath of coat proteins. Bacteriophage are very stable structures and billions of identical phage can be produced quickly and inexpensively from a bacterial culture. The goal of this project was to develop phage as preassembled components, larger than individual proteins, from which larger architectures could be built. To accomplish this goal, we employed techniques from molecular and synthetic biology to engineer phage to encode novel bio-interactive surface domains on their coat proteins. Approaches used included coiled-coil forming peptides, biotin tags (which bind streptavidin cross-linkers), and domains that auto-connect by forming intermolecular covalent bonds. In addition, we employed phage of different geometries, such as long filaments (M13) or icosahedral shells (T7), to diversify further our library of components, much like the linkers and hubs of a child’s construction set. Benchmarks in our project included (1) sequence confirmation of individual DNA constructs, (2) immunoblot assays to assess levels of display on engineered phage, (3) and the use of fluorescent markers to visualize the assembly of phage into pairwise dimers and higher order architectures. We also showed that environmentally-sensitive dyes could be linked to individual phage to generate nanosensors that could be incorporated into phage-based architectures to generate smart arrays report back on local conditions. Phage-based assembly represents a new approach to the development of novel architectures in biomaterials science. This project was carried out at Haverford College (Haverford, PA), a top-tier liberal arts college with a strong a tradition of involving undergraduate students in original research projects. The fourteen undergraduate students who participated in this RUI project are pursuing careers in research, medicine, teaching and biotechnology.
