CD
C.J.A. Danelon
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1
Inside and outside, intracellular and extracellular. This distinction is vital for cells and they expend vast amounts of energy to maintain the barriers that separate the in from the out. How this barrier, the cell envelope, is constructed varies wildly between organisms. We have long exploited differences between our own membrane and the bacterial cell envelope to develop targeted antibiotics. While this has resulted in a great deal of knowledge on bacterial, and especially Escherichia coli, cell envelope biosynthesis, many aspects of its regulation are still a mystery.
The phospholipid biosynthesis pathway is one the central pathways supplying crucial building blocks (phospholipids) to both the inner and outer membranes. In E. coli, much of this pathway has been thoroughly characterized and all the enzymes have been identified. Yet, despite decades of research, how this pathway is regulated remains disputed and unclear. Recent metabolomic research on the phospholipid and the preceding fatty acid biosynthesis pathways has now identified PlsB as the central point of regulation of both pathways. Furthermore, it was shown that its regulation is post-translational, and most likely through a negative feedback loop. PlsB is the first enzyme in the phospholipid pathway and catalyzes the committed step that sees the addition of acyl-ACP to glycerol- 3-phosphate. It has long been reported that PlsB is able to self-assemble into a filament, something that in other enzymes has been shown can act as a regulatory mechanism. Combining these advances we now seek to determine if filament formation is indeed how PlsB is regulated and if, as we suspect, the abundance of the membrane, its end product, is what controls the rate of filamentation..... ...
The phospholipid biosynthesis pathway is one the central pathways supplying crucial building blocks (phospholipids) to both the inner and outer membranes. In E. coli, much of this pathway has been thoroughly characterized and all the enzymes have been identified. Yet, despite decades of research, how this pathway is regulated remains disputed and unclear. Recent metabolomic research on the phospholipid and the preceding fatty acid biosynthesis pathways has now identified PlsB as the central point of regulation of both pathways. Furthermore, it was shown that its regulation is post-translational, and most likely through a negative feedback loop. PlsB is the first enzyme in the phospholipid pathway and catalyzes the committed step that sees the addition of acyl-ACP to glycerol- 3-phosphate. It has long been reported that PlsB is able to self-assemble into a filament, something that in other enzymes has been shown can act as a regulatory mechanism. Combining these advances we now seek to determine if filament formation is indeed how PlsB is regulated and if, as we suspect, the abundance of the membrane, its end product, is what controls the rate of filamentation..... ...
Inside and outside, intracellular and extracellular. This distinction is vital for cells and they expend vast amounts of energy to maintain the barriers that separate the in from the out. How this barrier, the cell envelope, is constructed varies wildly between organisms. We have long exploited differences between our own membrane and the bacterial cell envelope to develop targeted antibiotics. While this has resulted in a great deal of knowledge on bacterial, and especially Escherichia coli, cell envelope biosynthesis, many aspects of its regulation are still a mystery.
The phospholipid biosynthesis pathway is one the central pathways supplying crucial building blocks (phospholipids) to both the inner and outer membranes. In E. coli, much of this pathway has been thoroughly characterized and all the enzymes have been identified. Yet, despite decades of research, how this pathway is regulated remains disputed and unclear. Recent metabolomic research on the phospholipid and the preceding fatty acid biosynthesis pathways has now identified PlsB as the central point of regulation of both pathways. Furthermore, it was shown that its regulation is post-translational, and most likely through a negative feedback loop. PlsB is the first enzyme in the phospholipid pathway and catalyzes the committed step that sees the addition of acyl-ACP to glycerol- 3-phosphate. It has long been reported that PlsB is able to self-assemble into a filament, something that in other enzymes has been shown can act as a regulatory mechanism. Combining these advances we now seek to determine if filament formation is indeed how PlsB is regulated and if, as we suspect, the abundance of the membrane, its end product, is what controls the rate of filamentation.....
The phospholipid biosynthesis pathway is one the central pathways supplying crucial building blocks (phospholipids) to both the inner and outer membranes. In E. coli, much of this pathway has been thoroughly characterized and all the enzymes have been identified. Yet, despite decades of research, how this pathway is regulated remains disputed and unclear. Recent metabolomic research on the phospholipid and the preceding fatty acid biosynthesis pathways has now identified PlsB as the central point of regulation of both pathways. Furthermore, it was shown that its regulation is post-translational, and most likely through a negative feedback loop. PlsB is the first enzyme in the phospholipid pathway and catalyzes the committed step that sees the addition of acyl-ACP to glycerol- 3-phosphate. It has long been reported that PlsB is able to self-assemble into a filament, something that in other enzymes has been shown can act as a regulatory mechanism. Combining these advances we now seek to determine if filament formation is indeed how PlsB is regulated and if, as we suspect, the abundance of the membrane, its end product, is what controls the rate of filamentation.....
The research in this dissertation was conducted within the framework of the Building a Synthetic Cell (BaSyC) consortium, which aims to construct a minimal synthetic cell. This is a simple cell containing a minimal set of genes required and sufficient to exhibit the fundamental properties of life. Achieving this goal will deepen our understanding of the essential principles underlying cellular life. This dissertation explores a crucial step towards the realization of a minimal synthetic cell: the de novo design and assembly of its genome. Minimal genome assembly is performed by transformation of the yeast Saccharomyces cerevisiae with DNA fragments, which are assembled into a chromosome by the yeast’s native homologous recombination machinery. The assembled chromosomes are then isolated from yeast and tested for expression in vitro. With the methodologies developed in this dissertation for the design, assembly, screening, isolation, and characterization of synthetic chromosomes for the minimal cell, as well as the constructed chromosome prototypes, we have paved the way for future engineering of minimal genomes and integration of functional modules in synthetic cells.
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The research in this dissertation was conducted within the framework of the Building a Synthetic Cell (BaSyC) consortium, which aims to construct a minimal synthetic cell. This is a simple cell containing a minimal set of genes required and sufficient to exhibit the fundamental properties of life. Achieving this goal will deepen our understanding of the essential principles underlying cellular life. This dissertation explores a crucial step towards the realization of a minimal synthetic cell: the de novo design and assembly of its genome. Minimal genome assembly is performed by transformation of the yeast Saccharomyces cerevisiae with DNA fragments, which are assembled into a chromosome by the yeast’s native homologous recombination machinery. The assembled chromosomes are then isolated from yeast and tested for expression in vitro. With the methodologies developed in this dissertation for the design, assembly, screening, isolation, and characterization of synthetic chromosomes for the minimal cell, as well as the constructed chromosome prototypes, we have paved the way for future engineering of minimal genomes and integration of functional modules in synthetic cells.
Life, the most complex and admirable machine that one could think of has evolved over billions of years to display a beautiful variety of mechanisms that keep cells adapting, self-maintaining, reproducing, and evolving. If we think about it, what is this magic? What are the mechanisms behind life’s origins and wonderful coordination? Attracted by these intricates, different scientific disciplines have for long studied all life’s scales to grasp the fundamental principles of life. In particular, the synthetic biology field has set the goal of discerning life until the point that a minimal synthetic cell can be fully recreated in a controlled laboratory set-up. Synthetic cells, modular enough to be crafted by scientists, could not only reveal fundamental insights of how life works, but can also help unlock great biotechnological applications that lie beyond the reach of our current technologies and understanding of life. In this thesis, we delve into how in vitro evolution, module integration, and high throughput characterization are valuable steps to consider for accelerating the bottom-up assembly of artificial cells.
...
Life, the most complex and admirable machine that one could think of has evolved over billions of years to display a beautiful variety of mechanisms that keep cells adapting, self-maintaining, reproducing, and evolving. If we think about it, what is this magic? What are the mechanisms behind life’s origins and wonderful coordination? Attracted by these intricates, different scientific disciplines have for long studied all life’s scales to grasp the fundamental principles of life. In particular, the synthetic biology field has set the goal of discerning life until the point that a minimal synthetic cell can be fully recreated in a controlled laboratory set-up. Synthetic cells, modular enough to be crafted by scientists, could not only reveal fundamental insights of how life works, but can also help unlock great biotechnological applications that lie beyond the reach of our current technologies and understanding of life. In this thesis, we delve into how in vitro evolution, module integration, and high throughput characterization are valuable steps to consider for accelerating the bottom-up assembly of artificial cells.
Prior to cell division, a cell must generate an exact copy of its entire genome to ensure that each of the daughter cells obtains a full copy. This process, known as DNA replication, is an essential process of life and is vital for the health and survival of all cellular organisms.
In eukaryotes, DNA replication is catalyzed by a megadalton-sized dynamic protein complex known as the replisome, which generally carries out its important function in a remarkably efficient and accurate manner. This Herculean task becomes even more impressive when one considers the thousands of roadblocks along the way around which the replisome must navigate, such as tightly bound DNA-binding proteins, covalent DNA-protein crosslinks, highly stable DNA secondary structures, and several forms of DNA damage. In addition to replicating DNA, the replisome is also responsible for a critical process in epigenetic inheritance: the disassembly of nucleosomes on the parental DNA ahead of the replication fork, and their reassembly onto the newly synthesized DNA behind it. Finally, adding to the list of complex tasks it must achieve, the replisome is also able to sense some forms of DNA damage and facilitate their repair... ...
In eukaryotes, DNA replication is catalyzed by a megadalton-sized dynamic protein complex known as the replisome, which generally carries out its important function in a remarkably efficient and accurate manner. This Herculean task becomes even more impressive when one considers the thousands of roadblocks along the way around which the replisome must navigate, such as tightly bound DNA-binding proteins, covalent DNA-protein crosslinks, highly stable DNA secondary structures, and several forms of DNA damage. In addition to replicating DNA, the replisome is also responsible for a critical process in epigenetic inheritance: the disassembly of nucleosomes on the parental DNA ahead of the replication fork, and their reassembly onto the newly synthesized DNA behind it. Finally, adding to the list of complex tasks it must achieve, the replisome is also able to sense some forms of DNA damage and facilitate their repair... ...
Prior to cell division, a cell must generate an exact copy of its entire genome to ensure that each of the daughter cells obtains a full copy. This process, known as DNA replication, is an essential process of life and is vital for the health and survival of all cellular organisms.
In eukaryotes, DNA replication is catalyzed by a megadalton-sized dynamic protein complex known as the replisome, which generally carries out its important function in a remarkably efficient and accurate manner. This Herculean task becomes even more impressive when one considers the thousands of roadblocks along the way around which the replisome must navigate, such as tightly bound DNA-binding proteins, covalent DNA-protein crosslinks, highly stable DNA secondary structures, and several forms of DNA damage. In addition to replicating DNA, the replisome is also responsible for a critical process in epigenetic inheritance: the disassembly of nucleosomes on the parental DNA ahead of the replication fork, and their reassembly onto the newly synthesized DNA behind it. Finally, adding to the list of complex tasks it must achieve, the replisome is also able to sense some forms of DNA damage and facilitate their repair...
In eukaryotes, DNA replication is catalyzed by a megadalton-sized dynamic protein complex known as the replisome, which generally carries out its important function in a remarkably efficient and accurate manner. This Herculean task becomes even more impressive when one considers the thousands of roadblocks along the way around which the replisome must navigate, such as tightly bound DNA-binding proteins, covalent DNA-protein crosslinks, highly stable DNA secondary structures, and several forms of DNA damage. In addition to replicating DNA, the replisome is also responsible for a critical process in epigenetic inheritance: the disassembly of nucleosomes on the parental DNA ahead of the replication fork, and their reassembly onto the newly synthesized DNA behind it. Finally, adding to the list of complex tasks it must achieve, the replisome is also able to sense some forms of DNA damage and facilitate their repair...
Aquaporin-2 trafficking
Studying cellular mechanisms with subcellular aspiration and cryo-electron microscopy