From Genes to Gels: Programming the Physical and Biological Properties of Multifunctional Protein Hydrogels

ID：MPS/dmr/bmat(7623)1206121 PI：Tirrell，David ORG：Caltech标题：从基因到凝胶：编程多功能蛋白质水凝胶的物理和生物特性INTELLECTUCT优点：该项目使用重组DNA技术来制备多功能蛋白质生物材料。这种聚合物合成方法提供了对链长和单体序列的近乎绝对的控制，并使多功能材料的直接制备成为可能。蛋白质聚合物的物理化学性质可以通过开发将非规范氨基酸结合到蛋白质链中的方法来进一步工程。在这个项目中，利用蛋白质的生物合成来制备新的基于蛋白质的多功能蛋白质聚合物，这些聚合物可以组装成粘弹性凝胶。合成的材料被用于包裹和培养胰岛和胰岛，并将包含被认为对胰岛和胰岛生存至关重要的功能域。蛋白质的生物活性区域将主要由弹性蛋白衍生的序列组成；这些结构域将与亮氨酸拉链结构域交替，后者允许材料通过形成卷曲的聚集体组装成物理交联的水凝胶。这些凝胶将通过在环境条件下简单地混合蛋白质成分来实现细胞封装。弹性蛋白序列将被进一步定制，以包含非标准氨基酸叠氮高丙氨酸，这使得能够通过共价交联来控制粘弹性性质。这种方法利用菌种促进的叠氮-炔环加成反应温和的生物正交性来影响蛋白质凝胶的共价交联。这项工作的目标是(1)设计混合后凝胶的多域蛋白质，(2)使用温和的生物正交化学方法制备化学交联蛋白质水凝胶，以及(3)阐明包裹在物理和化学交联蛋白质水凝胶中的细胞的行为。更广泛的影响：该项目支持的研究将与一个新的高中科学计划相结合，该计划名为“基因到凝胶”，向学生介绍生物科学和材料科学之间的想法和研究。新项目将把从帕萨迪纳公立高中挑选出来的一小部分学生带到加州理工大学校园，进行为期三周的夏季动手实验室体验。该计划的一个重要内容将是学生的高中教师承诺在加州理工学院学生所做的基础上，开发演示或实验，供他们在课堂上使用。从“基因到凝胶”这个过程中的每一步，学生们都将获得实践经验。他们将培养携带编码亮氨酸拉链蛋白的人造基因的细菌菌株，学习如何表达和分离蛋白质，并通过凝胶电泳和质谱仪验证其分子量和纯度。这项工作中使用的蛋白质非常适合这类实验，因为它们可以很容易地通过循环通过较低的临界溶液温度来纯化。有了纯蛋白质，学生们将准备水凝胶，并通过反转测试、落球粘度计和简单的颗粒跟踪来表征相关的粘度变化。在计划结束时，所有参与者将共同选择和组织可以有效地转移到高中课堂上的活动。该项目将在整个项目过程中影响十几个教室和数百名高中生。

ID: MPS/DMR/BMAT(7623) 1206121 PI: Tirrell, David ORG: CaltechTitle: From Genes to Gels: Programming the Physical and Biological Properties of Multifunctional Protein HydrogelsINTELLECTUAL 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.