INTELLECTUAL MERIT: This project uses recombinant DNA technology to prepare multifunctional protein-based biomaterials. This approach to polymer synthesis provides near-absolute control over chain length and monomer sequence, and enables straightforward preparation of multifunctional materials. The physicochemical properties of protein polymers can be engineered further by exploiting methods for incorporating non-canonical amino acids into the protein chain. In this project, protein biosynthesis is harnessed to prepare new protein-based, multifunctional protein polymers that can be assembled into viscoelastic gels. The resulting materials are targeted for encapsulating and culturing pancreatic beta-cells and islets, and will contain functional domains thought to be important for beta-cell and islet survival. The bioactive regions of the proteins will be composed largely of elastin-derived sequences; these domains will alternate with leucine-zipper domains that permit assembly of the materials into physically crosslinked hydrogels through formation of coiled-coil aggregates. These gels will enable cell encapsulation through simple mixing of protein components under ambient conditions. The elastin sequences will be further tailored to contain the non-canonical amino acid azidohomoalanine, which enables control of viscoelastic properties through covalent crosslinking. This approach exploits the mild, bio-orthogonal character of the strain-promoted azide-alkyne cycloaddition to effect covalent crosslinking of protein gels. The goals of this work are (1) to engineer multidomain proteins that gel upon mixing, (2) to chemically crosslink protein hydrogels using mild, bio-orthogonal chemistries, and (3) to elucidate the behavior of cells encapsulated in physically and chemically crosslinked protein hydrogels.
BROADER IMPACTS: The research supported by this project will be integrated with a new high school science program, entitled "Genes to Gels," which introduces students to ideas and research at the interface between the biological sciences and materials science. The new program will bring to the Caltech campus a small number of students selected from Pasadena public high schools, for a three-week, summer hands-on laboratory experience. An important element of the program will be the commitment of the students' high school teachers to build on what the students do at Caltech, to develop demonstrations or experiments for use in their classrooms. Students will gain hands-on experience with each step of the process that takes us from "genes to gels." They will grow bacterial strains that carry artificial genes encoding leucine-zipper proteins, learn how to express and isolate proteins, and verify their molecular weights and purities by using gel electrophoresis and mass spectrometry. The proteins to be used in this work are well suited to experiments of this kind, because they can be purified easily by cycling through the lower critical solution temperature. With pure proteins in hand, students will prepare hydrogels and characterize the associated changes in viscosity by inversion tests, falling ball viscometry, and simple particle tracking. At the end of the program, all of the participants will work together to select and structure activities that can be productively transferred to the high school classroom. This program will impact a dozen classrooms and hundreds of high school students over the course of the project.
