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
University of Minnesota Ph.D. dissertation. August 2012. Major: Physics. Advisor: Vincent Noireaux. 1 computer file (PDF); vii, 107 pages.
Shin, G.Y.N Shin.
Molecular programming with a transcription and translation cell-free toolbox: from elementary gene circuits to phage synthesis..
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