Wasted Cells!
From vaccines to fuels and protein to plastic, cells have been harnessed to produce a variety of biomaterials! However, during downstream processing, extracting these products created from cells requires methods to kill the entire cell line, and these forms of disruption are often expensive.
Our team wondered if it would be possible to achieve this without cell death and thus increase the longevity of the cell line for continued production. Would it be possible to extract only the necessary amount from a cell (forcing exocytosis) and transport the desired product safely?
Inspiration From Inside
Luckily, a protective and flexible structure already exists within the cell – clathrin. This is a trimeric protein that forms a triskelion (three-legged) structure. The interaction of multiple triskelia results in a polyhedral cage-like structure that encapsulates vesicles.
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During the process of endocytosis, clathrin self-assembles into a lattice structure along the cytoplasm side of the plasma membrane. While there are many opposing hypotheses as to how the clathrin formation bends the membrane, generally, through reorganization of the lattice, the membrane folds in to create a vesicle. With the support of dynamin, the vesicle is then pinched off from the rest of the membrane, and the clathrin coated vesicle is delivered into the cytoplasm. [1]
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To get products from within the cell, we can utilize a similar method.
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Clathrin-coated pits forming on cell membrane [2].
Clathrin-mediated endocytosis process [3].
oxDNA trajectory of a single unit of MemBot [12,13].
Inspired by the clathrin triskelion, MemBot’s reconfigurable three-arm structure is built for rigidity and flexibility.
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In a similar fashion, individual MemBots can assemble on the outside of the cell membrane, pinching off with products contained within the vesicle. Not only does this preserve the original cell line, allowing continued division, but the clathrin-based skeleton adds an additional layer of protection during transport.
DNA
Origami
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Synthetic Cells
DNA origami was a technique that was first pioneered by Dr. Paul Rothemund in 2006 that utilized the programmability of DNA to create simple two-dimensional shapes [7]. Due to the versatility of this technique, it was later used to create higher order structures for applications in drug delivery, nanorobotics, and single molecule force measurements. Recent work has focused on the integration of DNA origami structures with synthetic cells to better study and understand discrete properties/mechanisms outside the complexity of the cell.
The integration of DNA origami has also been used to improve limitations with cell testing. Previous research has seen the use of DNA origami to create a nanoshell that improves viability and allows for multicellular assemblies as cell damage and death has been a limiting factor in cell printing and multicellular assemblies [1]. Along with integrating DNA origami to improve the viability of cells for testing, DNA origami has been used to create synthetic cells to isolate components and better understand mechanical and chemical interactions within the cell. Our research focuses on creating a building block out of DNA origami to create higher order assemblies that can be used for several different applications for synthetic cell testing.
Project
Goals
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Design the structure of MemBot with MagicDNA and caDNAno
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Complete initial folding and imaging of MemBot in the lab
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Optimize the folding process using a salt screen and annealing temperature gradient.
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Hierarchical assembly through linear polymerization
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​Achieve reconfigurability by performing angle change
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​Compared single stranded vs. double stranded connections to test flexibility
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Tested different types of double stranded connections
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Confirm success of structure through gel and TEM imaging
Future Applications
Synthetic Cell Membrane Application
Developing and researching artificial cells can achieve a variety of goals from understanding how life works to manufacturing drug molecules and detecting toxins, and unlike engineered cells, synthetic cells have the advantage of performing a single function to understand these processes in isolation.
While artificial cells can be developed from living cells (“top-down approach”), the process of generating cells from non-biotic components (“bottom-up approach) is more challenging. Out of the basic elements needed to replicate a cell, the semi-permeable membrane is one of the most crucial. While synthetic membranes have previously been constructed with polymers, proteins, and lipids, this approach has not yet been accomplished with DNA origami [5].
Previous uses of DNA origami have shown to increase the robustness of cell membranes. Most recently, Weitao Wang, et al. from Carnegie Mellon University, developed a DNA origami cell armor which was used to protect cells against mechanical stress [6]. Not only is this also a potential goal of our structure, but by incorporating flexibility to mimic endocytosis and potentially provide the capability to look into a cell to observe ongoing cell processes.
Contractile Ring Application
Contractile Rings are used to divide cells and often squeeze a cell to break it into two daughter cells. It is specifically used in cytokinesis, or cell division. The ring will contract around the parent cell and must pick the right point to begin pinching to ensure all cell DNA and organelles are divided equally between the two daughter cells. This contraction of the ring requires chemical energy, often in the form of adenosine triphosphate (ATP). The rings are made of actin, myosin, and other proteins and are primarily used in eukaryotic cell division.
Currently, Heidelberg University and Kerstin Göpfrich have developed a polymer ring that is considered a Contractile ring. This newly developed polymer ring is made of DNA nanotubes and works by using molecular attraction of parts of the ring to contract the ring into itself. The team uses one of two methods to cause the ring to contract. The first method uses “sticky ends” and has them attached to contract. The second method uses outside forces. The synthetic ring is surrounded by other molecules that force it into contracting. Neither of these methods require a chemical energy source.
Another synthetic contractile ring being researched, is one made of actin. Actin is one of the proteins naturally used in contractile rings. A uniquely developed “spatial positioning tool” to build these actins based contractile rings was designed. This specialized tool was created from the bacterial MinDE protein system by in-vitro methods. Using this approach, allows the synthetic actin ring to be built around the middle of the cell of interest by active transport. This method is being studied in the division of giant unilamellar vesicles (GUVs).