In vitro informational approach to biopolymer network dynamics

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.