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：编程多功能蛋白水凝胶的物理和生物学特性智力优点：该项目使用重组DNA技术制备多功能蛋白基生物材料。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，并且可以直接制备多功能材料。通过开发将非规范氨基酸纳入蛋白质链的方法，可以进一步设计蛋白质聚合物的物理化学性质。在这个项目中，蛋白质生物合成被用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。所得到的材料是针对包封和培养胰腺β细胞和胰岛，并将包含功能域被认为是重要的β细胞和胰岛的生存。蛋白质的生物活性区将主要由弹性蛋白衍生序列组成；这些结构域将与亮氨酸-拉链结构域交替存在，亮氨酸-拉链结构域允许通过形成盘绕式聚集体将材料组装成物理交联的水凝胶。这些凝胶可以在环境条件下通过简单的混合蛋白质成分来实现细胞封装。弹性蛋白序列将进一步定制，以包含非规范氨基酸叠氮同质丙氨酸，这使得通过共价交联控制粘弹性特性成为可能。该方法利用菌株促进叠氮化物-炔环加成反应温和的生物正交特性来影响蛋白凝胶的共价交联。这项工作的目标是：(1)设计混合后凝胶化的多结构域蛋白质，(2)使用温和的生物正交化学方法化学交联蛋白质水凝胶，(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名为“从基因到凝胶”的新高中科学计划相结合，该计划向学生介绍生物科学和材料科学之间的界面的想法和研究。这个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学院校园，进行为期三周的暑期动手实验体验。这个项目的一个重要组成部分将是学生的高中老师的承诺，他们将以学生在加州理工学院所做的为基础，开发示范或实验，用于他们的课堂。学生将获得从“基因到凝胶”过程的每一步的实践经验。他们将培养携带编码亮氨酸拉链蛋白的人工基因的细菌菌株，学习如何表达和分离蛋白质，并通过凝胶电泳和质谱法验证它们的分子量和纯度。在这项工作中使用的蛋白质非常适合这种实验，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纯化。有了纯蛋白质，学生将制备水凝胶，并通过倒置测试、落球粘度法和简单的颗粒跟踪来表征相关的粘度变化。在项目结束时，所有参与者将共同选择和组织能够有效转移到高中课堂的活动。在整个项目过程中，该项目将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.