Molecular Programming with a Transcription and Translation Cell-Free Toolbox: From Elementary Gene Circuits to Phage Synthesis

Cell-free synthetic/systems biology is an emerging field connecting biology, chemistry, physics, and engineering to understand biological systems and expand their capabilities. 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 system 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 transcriptional 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.