Molecular programming with a transcription and translation cell-free toolbox: from elementary gene circuits to phage synthesis.

Abstract

Cell-free synthetic/systems biology is an emerging field connecting biology, chem- istry, physics, and engineering to understand biological systems and expand their capa- bilities. In vitro approaches compared to in vivo allow much better control of parameters and give much more freedom to program and study biological systems. Among the in vitro approaches, a transcription and translation (TX-TL) cell-free gene expression sys- tem mimicking a natural biological system offers the closest context to an intact cell. The conventional cell-free system as a playground to perform an experiment, however, has a couple of serious problems such as an insufficient sink system and the lack of transcrip- tional diversity. In this dissertation, I report the preparation of a custom-made E. coli cell-free system for the purpose of quantitative synthetic/systems biology, demonstrate synthetic gene circuits with cell-free toolbox, and show cell-free synthesis of a functional entity from genome-sized DNA. The custom-made cell-free system expresses genes with only endogenous TX-TL machinery and the sink systems for two biomolecules, mRNA and protein, can be applied in it. Moreover, mathematical models of gene expression including sink systems in this cell-free system are described. As a concept of cell-free toolbox, this cell-free system also makes it possible to use a variety of transcriptional activation and repression units to construct elementary circuit motifs. Furthermore, a bacteriophage as complex as T7 phage is synthesized from its genome-sized DNA with this cell-free system. This cell-free synthesis in a single test tube includes the central dogma of molecular biology including transcription, translation, and DNA replication as an internal process, and self-assembly and DNA packaging as a post-gene-expression process.