Migas, Urszula Magdalena (2016) A Bottom-Up Approach to Building an Artificial Cell by Self-Assembly. PhD thesis, National University of Ireland Maynooth.

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Abstract

Synthetic biology is a rapidly growing field in which recent advances now allow the formation of minimal or artificial cells composed of a minimum number of components, capable of performing specific functions. New developments contributing to the complexity of artificial cells or making their design more cell-like increases the number of possible applications. To-date many processes and chemical reactions have been studied in these cells; many more remain to be explored. However, more sophisticated approaches to artificial cell design and preparation will be required to do this. The results presented here provide insights into how artificial cell development can contribute to our understanding of the self-assembly of biomolecules. The formation of lipid based vesicles is an inherent element of artificial cell development, which requires reliable techniques to prepare vesicles of cell size. The most widely applied methods have been evaluated here based on the size, quality and abundance of vesicles formed as well as the ease of encapsulating biological solutes. The effect of various lipid compositions, particularly cholesterol, has also been analysed. This comparison provides reliable information for tailoring the selection of experimental approach when building a model cell. Functionalisation of the artificial cell surface and its interior is required for many applications. For surface modification, there is growing interest in using glycolipids to fulfil a molecular recognition role. A synthetic glycolipid has been incorporated into the phospholipid membrane of giant unilamellar vesicles at biologically relevant concentrations. The synthetic glycolipid shows concentration-dependent phase behaviour in binary mixtures with DOPC and in ternary mixtures with DOPC and cholesterol. At low concentrations, the glycolipid is fully dispersed in the GUV membrane. At concentrations above 10%, the formation of lipid tubules was observed, consistent with the formation of a columnar lipid phase. Lipid tubules are observed in aqueous and oil solvents, suggesting that both hexagonal and inverted hexagonal lipid arrangements can be formed. The self- assembly of proteins in cells is required for normal biological function and unintended self-assembly also can occur following a change in environmental conditions, in some cases leading to disease. To understand these processes more fully, experiments performed in controlled, but closer to physiological conditions are required. Artificial cells provide an idea platform to do this. Bovine Serum Albumin (BSA) was encapsulated in phospholipid based giant unilamellar vesicles of cell size. The formation of aggregates within the GUV was analysed using a fluorescent dye (Thioflavin T) and various modes of microscopy. While protein aggregation was observed inside the vesicles, harsh environmental conditions were required to induce this aggregation (e.g.heat, low pH). An approach to investigate protein condensation upon in-situ expression in physiologically relevant conditions was also explored. In this case, a protein that aggregates at physiological temperature and pH (the P23T mutant of human gamma D crystallin) was expressed in-situ inside a GUV using a cell-free expression system. The formation of P23T specific aggregates was observed after incubation for several hours at 37°C. These aggregates have a fractal dimension lower than those normally observed for amorphous protein aggregate. Furthermore, we have demonstrated that the self-assembly of P23T can also be induced following transfection in mammalian cells, providing deeper insights into the mechanism by which the genetic cataract associated with this mutation occurs.

Item Type:	Thesis (PhD)
Keywords:	Bottom-Up Approach; Building; Artificial Cell; Self-Assembly;
Academic Unit:	Faculty of Science and Engineering > Chemistry
Item ID:	10412
Depositing User:	IR eTheses
Date Deposited:	09 Jan 2019 10:18
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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