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Microfabrication of cell-like 3D geometry for in vitro reconstitution of dynamic protein systems
Microfabrication of cell-like 3D geometry for in vitro reconstitution of dynamic protein systems
The creation of in vitro environments that mimic cellular conditions is a key part of bottom-up synthetic biology. Such environments must contain relevant biochemical and biophysical functionalities, as well as the right kind of shape - as geometry plays a crucial role in many biological processes. In this regard, microfabrication is an effective way to pattern structures on length scales that can influence protein function. With emerging technologies in biochemical and fabrication tools, we face a continuous challenge to adapt to new methods, and then to innovate on design ideas that would give us a better understanding of the biological system in question. In this thesis, I focused on developing tools with which we can modulate the geometry of in vitro environments that involve, in particular, lipid membranes. The lipid interface is a crucial component of the cellular environment, where many protein functions take place. However, reliably controlling their morphology is an issue that has not yet been conclusively solved. In a series of projects, I developed ways to pattern supported lipid membranes on rigid supports, in dimensions ranging from planar 2D substrates, embossed 2.5D compartments, and fully 3D structures. These were in turn used to interrogate the geometry sensitivity of the E. coli MinDE system. This protein system forms a reaction-diffusion network, which self-organise into dynamic patterns based on an intricate interplay between their diffusion between the bulk and the membrane, as well as their associated reaction kinetics. Therefore, the membrane geometry plays a crucial role in their pattern formation, and thus became a target of deeper investigation. In a quest to then create free-standing membrane geometries that mimic cellular shapes, I demonstrated the fabrication of dynamic, shape-shifting 4D structures made of protein-based hydrogels that deform lipid vesicles. I also investigated the reengineering of more stable free-standing membrane systems from synthesized dendrimer molecules that permit more strenuous shape transformations. By recapitulating MinDE patterns on these synthetic membranes, we demonstrated their functional analogy to the lipid bilayer, thus proposing an alternative model membrane system for synthetic biology. Finally, I applied 3D printing to enable the upright imaging of bacteria, facilitating the imaging of the divisome by super-resolution techniques. While these investigations primarily revolve around the study of the Min system, the methods developed in this thesis have wider applications in synthetic biology in general, where membrane interacting or associated proteins with geometry sensitive properties are concerned. The 3D printing of dynamic, shape-transforming structures also have implications beyond membranous systems, where we can anticipate further developments that build on this technology to recapitulate more complex, biomimetic behaviours. Thus, this thesis presents exciting methodological advancements for bottom-up synthetic biology.
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Eto, Hiromune
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Eto, Hiromune (2021): Microfabrication of cell-like 3D geometry for in vitro reconstitution of dynamic protein systems. Dissertation, LMU München: Faculty of Physics
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Abstract

The creation of in vitro environments that mimic cellular conditions is a key part of bottom-up synthetic biology. Such environments must contain relevant biochemical and biophysical functionalities, as well as the right kind of shape - as geometry plays a crucial role in many biological processes. In this regard, microfabrication is an effective way to pattern structures on length scales that can influence protein function. With emerging technologies in biochemical and fabrication tools, we face a continuous challenge to adapt to new methods, and then to innovate on design ideas that would give us a better understanding of the biological system in question. In this thesis, I focused on developing tools with which we can modulate the geometry of in vitro environments that involve, in particular, lipid membranes. The lipid interface is a crucial component of the cellular environment, where many protein functions take place. However, reliably controlling their morphology is an issue that has not yet been conclusively solved. In a series of projects, I developed ways to pattern supported lipid membranes on rigid supports, in dimensions ranging from planar 2D substrates, embossed 2.5D compartments, and fully 3D structures. These were in turn used to interrogate the geometry sensitivity of the E. coli MinDE system. This protein system forms a reaction-diffusion network, which self-organise into dynamic patterns based on an intricate interplay between their diffusion between the bulk and the membrane, as well as their associated reaction kinetics. Therefore, the membrane geometry plays a crucial role in their pattern formation, and thus became a target of deeper investigation. In a quest to then create free-standing membrane geometries that mimic cellular shapes, I demonstrated the fabrication of dynamic, shape-shifting 4D structures made of protein-based hydrogels that deform lipid vesicles. I also investigated the reengineering of more stable free-standing membrane systems from synthesized dendrimer molecules that permit more strenuous shape transformations. By recapitulating MinDE patterns on these synthetic membranes, we demonstrated their functional analogy to the lipid bilayer, thus proposing an alternative model membrane system for synthetic biology. Finally, I applied 3D printing to enable the upright imaging of bacteria, facilitating the imaging of the divisome by super-resolution techniques. While these investigations primarily revolve around the study of the Min system, the methods developed in this thesis have wider applications in synthetic biology in general, where membrane interacting or associated proteins with geometry sensitive properties are concerned. The 3D printing of dynamic, shape-transforming structures also have implications beyond membranous systems, where we can anticipate further developments that build on this technology to recapitulate more complex, biomimetic behaviours. Thus, this thesis presents exciting methodological advancements for bottom-up synthetic biology.