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.
Cell-free protein synthesis was developed in the 60s to elucidate the genetic code. DNA-dependent cell-free expression systems became widely used as a research tool in the late 60s and the 70s to analyze gene products and to understand the regulation of gene expression such as the E. coli lactose and tryptophan operons. The development of highly efficient hybrid cell-free expression systems in the early 90s was a turning point for this technology. The modern transcription-translation (TX-TL) cell-free systems are optimized for large-scale protein synthesis as an alternative to the recombinant protein technology. Commercially available cell-free TX-TL systems are used in an increasing number of applications in biotechnology, industry and proteomics. TX-TL cell-free TX-TL systems could also become powerful synthetic biology platforms to construct complex biochemical systems using a bottom-up approach. The construction of biological systems in a test tube using DNA programs provides a means to study biochemical behaviors in isolation, with a greater level of control and a greater freedom of design compared to in vivo. Constructing information-based biochemical systems in vitro offers the possibility of expanding the capabilities of existing biological systems. Elementary gene circuits, pattern formation and prototypes of artificial cells have been engineered with TX-TL cell-free systems. However, the development and the quantitative investigation of such complex systems in a cell-free context are limited by the current available technology, which has not been optimized to construct information-based molecular systems. The expression of genome-sized DNA programs that recapitulates the entire chain of biological information, with the circuitry and the self-organization of complex active biological entities has not been demonstrated in a cell-free context. During the last five years, my laboratory has developed a custom-made cell-free transcription-translation (TX-TL) system to construct complex biochemical systems in vitro such as regulatory gene networks and self-assembled systems. This unique platform was optimized for cell-free synthetic biology applications. Cell-free gene expression is carried out by the endogenous transcription and translation machineries of E. coli. Protein synthesis is as efficient as the commercially available systems. Our system was also developed so as to control the global mRNA degradation rate and the degradation of proteins without damaging the transcription and the translation machineries. We have constructed a transcription repertoire of regulatory elements that works like the main transcription scheme of E. coli. The endogenous holoenzyme E70 is used as the housekeeping transcription machinery. The six other sigma factors, the bacteriophage RNA polymerases T7 and T3, and a set of repressors can be expressed to construct various types of circuits, such as transcriptional activation cascades (Figure 1A and 1B), negative feedback loops and combinations of both. Recently, we were able to cell-free synthesize two bacteriophages from their entire genome: the coliphages phiX174 and T7 composed 12 and 60 genes respectively. The bacteriophage T7 is not only entirely re-expressed in vitro, its DNA genome is also replicated (Figure 1C). We have demonstrated that our custome made cell-free TX-TL system can recapitulate the entire chain of biological information: transcription, translation, DNA replication and self-assembly of a fucntional complex biological entity. We are now trying to use this cell-free platform to construct an artificial cell. The cell-free TX-TL systems is encapsulated inside synthetic phospholipid vesicles which are programmed with gene circuits (Figure 1D). In one of our last work, we have shown how a cytoskelton can be polymerized at the inner membrane of the artifical cells. Eight articles related to this work have been published between 2010 and 2012.
