In vitro informational approach to biopolymer network dynamics

在本提案中，PI将研究体外生物聚合物网络的组装。PI方法耦合信息流和肌动蛋白细胞骨架结构的形成和动力学，它提供了材料科学和生物技术的前景。E.大肠杆菌无细胞表达系统已被改造成具有新的特性，包括：控制克隆基因的表达;使用三种不同的RNA聚合酶和三种不同的转录因子及其各自的启动子/操纵子文库;对转录物和蛋白质降解进行微调;以及三种表达形式：分批、乳液和连续模式的囊泡。该工具将允许PI研究细胞骨架相关机制的动力学，包括完整的信息链，细胞的格式和大小以及延长的时间段。在该项目的第一部分，将完成这种多功能表达系统的表征。基因网络将被工程化以及时定量和准确地控制基因的表达。这项工作将用于该项目的第二部分，通过表达肌动蛋白结合和聚合蛋白质，在二维无限空间中诱导肌动蛋白网络的形成。肌动蛋白丝的表达、扩散和交联网络结构之间的关系将被研究。最后，肌动蛋白网络将在合成囊泡的内膜处聚合。研究空间对称性破缺、突起形成和力产生的机制。该项目的主要智力价值是开发了一种创新方法，以定量研究生物聚合物网络组装和动力学的信息和物理特性。该研究方法将为肌动蛋白结构的细胞生物学提供定量的见解。该项目的更广泛影响的一部分包括对生物物理学本科生和研究生的培训。学生们将在实验室中进行实践。实验范围从标准克隆到理解活细胞中的模式形成和力的产生。学生还将参与基因电路和扩散机制的模型和模拟的开发。这项工作将对生物物理学和生物工程学等多个科学领域产生影响。该方法还将为合成，系统和细胞生物学做出重大贡献。

In this proposal the PI will study the assembly of biopolymer networks in vitro. The PIs approach couples information flow and actin cytoskeleton structures formation and dynamics, and it provides perspectives in material science and biotechnology. E. coli cell-free expression system has been engineered with new properties that include: the control of expression from cloned genes; the use of three different RNA polymerases and three different transcription factors with their respective promoters/operators libraries; the fine tuning of transcript and protein degradation; and three formats of expression: batch, emulsion, and vesicle in continuous mode. This tool will allow the PI to study the dynamics of cytoskeleton related mechanisms with the complete chain of information, in the format and size of the cell as well as over extended period of time. In the first part of the project, characterization of this versatile expression system will be completed. A gene networks will be engineered to control quantitatively and accurately the expression of genes in time. This work will be used in the second part of the project to induce actin networks formation in a two dimensional unlimited space by expressing actin binding and polymerizing proteins. The relationship between expression, diffusion and structure of crosslinked networks of actin filaments will be studied. Finally, actin networks will be polymerized at the inner membrane of synthetic vesicles. Mechanisms of space symmetry breaking, protrusion formation and force generation will be investigated. The main intellectual merit of the project is the development of an innovative method to study quantitatively the information and physics properties of biopolymer networks assembly and dynamics. The research approach will provide quantitative insights into cell biology of actin structures. Part of the broader impact of this project includes the training of undergraduate and graduate students in biophysics. The students will have a hands-on experience in the laboratory. The experiments will range from standard cloning to the understanding of pattern formation and force generation in living cells. Students will be also involved in the development of models and simulations of gene circuits and diffusion mechanisms. This work will have an impact on a variety of scientific fields, in biological physics and bioengineering. The approach will also make significant contributions to synthetic, systems and cell biology.