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WIREs Nanomed Nanobiotechnol
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The engineering of artificial cellular nanosystems using synthetic biology approaches

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Artificial cellular systems are minimal systems that mimic certain properties of natural cells, including signaling pathways, membranes, and metabolic pathways. These artificial cells (or protocells) can be constructed following a synthetic biology approach by assembling biomembranes, synthetic gene circuits, and cell‐free expression systems. As artificial cells are built from bottom‐up using minimal and a defined number of components, they are more amenable to predictive mathematical modeling and engineered controls when compared with natural cells. Indeed, artificial cells have been implemented as drug delivery machineries and in situ protein expression systems. Furthermore, artificial cells have been used as biomimetic systems to unveil new insights into functions of natural cells, which are otherwise difficult to investigate owing to their inherent complexity. It is our vision that the development of artificial cells would bring forth parallel advancements in synthetic biology, cell‐free systems, and in vitro systems biology. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
The mutual impacts of research in artificial cells and synthetic biology. Artificial cells are constructed by encapsulating synthetic gene circuits (the information) and expression systems (the engine) inside membranes (the shell). These artificial cells have been used as biomimetic systems for biological studies and as in situ expression systems for biotechnological applications. Furthermore, we envision that the development of artificial cells would bring forth parallel advancements of each of the subcomponents. Specifically, the engineering of membranes for artificial cells could lead to new insights into cellular division and membrane–protein interactions. The optimization of cell‐free systems for artificial cells could generate new findings into molecular crowding, protein–protein interactions, and ribosome biogenesis. Finally, the engineering of gene circuits in cell‐free systems could unveil new insights into dynamics of gene expression and establish a new platform for fast characterization of synthetic gene circuits. (Reprinted with permission from Ref 10. Copyright 2013 Nature Publishing Group; Reprinted with permission from Ref 29. Copyright 2013 Nature Publishing Group)
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The information: applications of genetic modules in artificial cellular systems. (a) A high crowding condition (open bars) reduced fold perturbation of gene expression when compared to a low crowding condition (gray bars). Chemicals were added to perturb gene expression. (Reprinted with permission from Ref 10. Copyright 2013 Nature Publishing Group). (b) Single‐molecule imaging was used to study the impact of molecular crowding on T7 RNAP binding to DNA promoter. (Reprinted with permission from Ref 10. Copyright 2013 Nature Publishing Group). (c) Fabrication of a 20 femtoliter nano‐chamber for the study of gene expression using cell‐free expression systems. (Reprinted with permission from Ref 92. Copyright 2013 American Chemical Society) (d) A delayed gene circuit was used to create oscillations in cell‐free systems. T3 RNAP activated its own expression (top panel). T3 RNAP also activated the expression of SupD that activated the expression of TetR. TetR then repressed the expression of T3 RNAP. The circuit generated sustained oscillations in a cell‐free system (bottom panel). (Reprinted with permission from Ref 93. Copyright 2013 The National Academy of Sciences)
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The information: the incorporation of genetic modules inside artificial cells. (a) A schematic of R3C ribozyme self‐replication. A duplex of R3C ribozyme (T.T) dissociated into two RNAs (T). RNA T‐induced ligation of two substrate RNAs A and B through a complex of RNA (A.B.T). A new duplex of R3C ribozyme (T.T) formed after the ligation. Reprinted with permission from Ref 83. Copyright 2002 The National Academy of Sciences (b) A template‐directed synthesis of RNA was reconstituted inside protocells that consisted of fatty‐acids membranes. Nucleotides from the extracellular environment diffused into the protocells to support the RNA synthesis. (Reprinted with permission from Ref 18. Copyright 2008 Nature Publishing Group). (c) A two‐level genetic cascade was reconstituted inside artificial cells. Output luminescence signals (filled circles) increased with time and exhibited a time delay due to the gene cascade. (Reprinted with permission from Ref 84. Copyright 2003 The National Academy of Sciences). (d) A two‐level genetic cascade was encapsulated inside artificial cells. A flow cytometer was used to measure gene expression rates inside artificial cells. The results show that artificial cells expressed green fluorescent proteins as an output (left panel). The negative control without DNA did not generate GFP (right panel). (Reprinted with permission from Ref 16. Copyright 2004 Elsevier).
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The engine: the impact of genetic and nongenetic factors on cell‐free expression systems. (a) The impact of molecular crowding on gene expression. Systems with a small crowder (left) showed a biphasic shape in gene expression with increasing crowding densities. Systems with a big crowder (right) exhibited monotonic increase of gene expression with increasing crowding densities. Weak genetic components resulted in faster increase in normalized gene expression rates when compared to a wild type component. PT7,weak represents a weak T7 promoter. RBSweak represents a weak ribosomal binding site. (Reprinted with permission from Ref 10. Copyright 2013 Nature Publishing Group). (b) Mapping of interactions between a protein synthesis system and ORF products. ORFs in the inner circle directly affected the minimal system, while those in the outer circle did not affect protein synthesis. Green circles represent proteins that caused beneficial effects. Yellow circles represent proteins that caused deleterious effects. (Reprinted with permission from Ref 22. Copyright 2008 The American Society for Biochemistry and Molecular Biology). (c) Expression of target proteins in microarrays. Eight different DNAs encoding target proteins fused to glutathione S‐transferase (GST) tag were immobilized in microarray format on a glass slide. Cell‐free systems were added to carry out the protein synthesis. The protein expression was confirmed using GST antibodies. (Reprinted with permission from Ref 73. Copyright 2004 The American Association of the Advancement of Science)
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The engine: the modulation of protein synthesis rates in cell‐free systems. (a) Correlation of ATP (panel 1) and protein (panel 2) concentrations with (II) or without (I) ATP regeneration. Spheres represent ATP; squares represent ADP; triangles represent AMP. Without ATP regeneration, the concentration of ATP declined rapidly in 4 h and dihydrofolate reductase (DHFR) synthesis almost stopped. With regeneration (supplemented with dithiothreitol), the decrease of ATP concentration was slowed and DHFR synthesis was prolonged. Reprinted with permission from Ref 25. Copyright 1994 Japan Society for Bioscience, Biotechnology and Agrochemistry (b) Synthesis of enhance GFP (eGFP) in liposomes using a cell‐free system. E. coli extract and plasmid encoding eGFP were encapsulated in a vesicle (left) and a doublet (right). eGFP expression was detected using fluorescence imaging. The scale bar represents 15 µm. (Reprinted with permission from Ref 38. Copyright 2004 The National Academy of Sciences). (c) eGFP expression in vesicles with (right) or without (left) α‐hemolysin. With α‐hemolysin, protein synthesis was prolonged significantly to over 100 h, which was much longer than without α‐hemolysin. The inset represents GFP intensities in the first 10 h. Filled spheres (right) represent eGFP expression with α‐hemolysin; filled squares (right) represent eGFP expression without α‐hemolysin. (Reprinted with permission from Ref 38. Copyright 2004 The National Academy of Sciences)
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The shell: The modulation of membrane dynamics. (a) Modeling of a late stage of exocytosis using liposomes connected to nanotubes. (1–4) A small liposome grew with the injection of fluid. The nanotube changed its shape from cylindrical to toroidal and the enclosed materials were released. After one round of stimulation, a new nanotube was formed. (5–8) Visualization of exocytosis using fluorescein‐filled vesicles. Fluorescence images 5–8 correspond to the events in 1–4. The scale bar represents 10 µm. (Reprinted with permission from Ref 23. Copyright 2003 The National Academy of Sciences). (B) Electrofusion and budding formation of vesicles containing macromolecules. Voltage supply was turned on or off at the top and bottom sides of a chamber that contained the vesicles. Vesicles started to fuse at time 0 s (indicated by white arrows). The fusion was completed after 20s. The budding transformation started at 42 s (indicated by a gray arrow). Budding shape was observed after 63 s. The scale bar represents 10 µm. (Reprinted with permission from Ref 43. Copyright 2012 The National Academy of Sciences)
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The shell: stability and permeability of artificial cellular membranes. (a) The concentrations of nonanoic acid micelles with (filled circles) and without (filled squares) nonanol were estimated by their absorbance under various pH conditions. The slope of the curves corresponds to a transition from micelles to droplets. Mixing of the fatty acid and alcohol remarkably slowed the transition and stabilized the vesicles under pH changes. (Reprinted with permission from Ref 31. Copyright 2002 Elsevier). (b) Relative permeability of liposomes to ribose with various membrane compositions, myristoleic acid (MA, C14:1), lauric acid (LA, C12:0), farnesol, oleate acid (OA, C18:1), and linoleate (C18:2). MA displayed higher permeability than longer fatty acid (OA). Linoleate liposomes were more permeable than OA liposomes that had the same length, but with a higher degree of saturation. Mixing of farnesol with MA increased fluidity and yielded higher permeability relative to pure MA. In contrast, the addition of LA in MA decreased fluidity and lowered the permeability. (Reprinted with permission from Ref 18. Copyright 2008 Nature Publishing Group)
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