The past decades have seen the rapid development of many aspects of synthetic biology. For example, attempts to build synthetic cells under controlled conditions in the laboratory have led to significant achievements. Following a bottom-up approach, scientists aim at building a self-reproducing synthetic cell with a minimum number of biological modules. A functional synthetic cell should accomplish at least four processes during one cycle: growth,DNAreplication,DNA segregation, and division. DNA segregation, as a vital step in the life cycle of a synthetic cell, is in focus in this thesis. Segregation of replicated DNA in eukaryotes depends highly on microtubules. Microtubules are protein polymers which form cylindrical tubes, a dynamic structure with which they can exert both pulling and pushing forces. During cell division, microtubules form a spindle-like structure, named the mitotic spindle. Microtubules in the mitotic spindle apparatus attach to the replicated chromosomes through kinetochores and exert pulling forces to separate the sister chromosomes and place them in the newly born daughter cells. These hollow tubes can grow in the presence of GTPbound tubulin dimers. When all the GTP-tubulin subunits at the end of a filament turned into GDP-tubulin, themicrotubule exhibits a transition to shrinkage. While the addition of GTP-tubulin to the end of microtubulesmay cause pushing forces, shrinking microtubules are able to exert pulling forces with the help of microtubule adapter proteins. The random transitions between growth and shrinkage of a microtubule is called dynamic instability and has been studied throughout decades. DNA segregation components of a synthetic cell should preferably be fully expressible in a cell-free manner which employs reconstituted transcription-translation factors. Although mitosis in eukaryotes is the best-studied DNA segregation system to date, microtubules are unsuitable for cell-free expression due to their complex chaperondependent folding as well as essential post-translational modifications. Interesting alternatives for DNA segregation in synthetic cells are provided by bacterial segregation systems. An active bacterial segregation system is ideally composed of three elements: a cytomotive NTPase filament, a connecting or adapter protein, and a centromere-like DNA sequence. This thesis will examine various bacterial cytoskeletal filaments with a central focus on their suitability for building a DNA segregation systemfor synthetic cells...@en

The last few years have shown promising developments in single-molecule protein sequencing using a biological nanopore. Using only one membrane pore in a salt buffer, an applied potential creates a measurable ion current through the pore’s constriction. If a small molecule passes through the pore, this disruption is visible as a characteristic current pattern based on the molecular structure of the molecule. Recent developments by Henry in 2021 show that single amino acid substitution in short proteins can be detected using this method. Unfortunately, due to the complexity of the reading, de novo sequencing does not yet apply to these single molecule reads. Nevertheless, detecting different variants of a protein is possible. In current work this method is being developed to detect phosphorylation sites on a peptide of biological significance. For the latter, an immunopeptide called IRS2 is chosen to be once and twice phosphorylated. This phosphorylation causes the peptide to be a cancer biomarker. If this low-cost method of post-translational modification detection is successful, it would be an improvement compared to the standard mass spectrometry sequencing approach. In this BEP research, problems faced before and after sequencing the IRS2 peptide are approached from two different perspectives. The first aim is to adapt the data acquisition software to automate the workflow. The second aim is to adapt and model the Freely Jointed Chain (FJC) Model to a heterogeneously charged peptide. Both aims are defined to be applicable to other peptides as well. For the first aim, a LabVIEW plugin was developed using insights from data processing in MATLAB. This tool detects the state of the nanopore reading, responds using voltage control in a closed feedback loop and frequently allows for calibration checks. All features were tested with training sequences from real data and show promising results. However, more testing in the lab is required to determine its accuracy compared to a human operator. For the second aim, the FJC model was analytically adapted to the IRS2 peptide inside the nanopore’s electric field. The energetically most favourable configuration was then sought with a Metropolis Algorithm. As a result, the electrostatic potential was calculated and implemented into the Metropolis Algorithm. Despite the simplification of this method, it is still expandable in a modular way to incorporate additional potential such as charge-charge interactions or springs to simulate backbone flexibility. Finally, improving this model further would eventually lead to the relative positions of all chain elements inside the pore to develop an understanding of the (phosphorylated) IRS2 readings.

Cells, the building blocks of life, are vastly complex. This complexity confers to every living organism the ability to maintain oneself, reproduce oneself, and evolve. Creating a minimal system from nonliving components that is capable of self-maintenance, self-reproduction, and evolvability, will greatly increase our understanding of life. Essential features of every cell, synthetic or otherwise, are the compartment, a form of information transfer, and the ability to proliferate. In chapter 1, I argue that autonomy, that is self-governance, is another key characteristic of life. Therefore, to create a synthetic cell, aforementioned features should be recapitulated in an autonomous manner. This informs the synthetic cell design that is adhered to in this thesis. Phospholipid vesicles, so called liposomes, serve as a compartment. Reconstitution of the central dogma of molecular biology by cell-free gene expression with the PURE system enables the use of a genetic program encoded on DNA. By encoding its working instructions on its own DNA, the cell will be autonomous. Proliferation is carried out by a set of modules encoded on the DNA. These modules will serve to replicate the DNA itself, to replenish the gene expression machinery, and to stimulate growth and trigger division of the compartment. The goal of this thesis is to reconstitute compartment growth by cell-free gene expression of DNA encoding for phospholipid synthesis machinery.@en

Synthetic biology is an emerging and rapidly expanding field of research focused on the assembly of novel biological systems with new functionalities tailored for different applications. Genetic circuits have been re-wired or constructed with elements from different organisms, and metabolic pathways have been engineered to endow cells with non-natural capabilities. One of the most exciting goals of synthetic biology is bringing solutions to biomedical challenges. Though this research area is still in its infancy, translational medicine is already witnessing the first steps towards the development of therapies based on synthetic biology. Cell-free synthetic biology (or in vitro synthetic biology), a branch of synthetic biology, makes use of cell-free gene expression systems to create biological networks that operate outside the chassis of a living cell. More than a decade ago, the convergence of synthetic biology, cell-free gene expression systems and liposome technology gave rise to the creation of artificial vesicle bioreactors that can synthesize genetically-encoded molecules. Though the primary motivation of these studies was the assembly of a semi-synthetic cell, the application of such technologies for different therapeutic purposes has been envisioned. Examples of this are the creation of antigen-producing liposomes as novel vaccination systems, the development of bioreactor liposomes suited for remotely controlled in-situ mRNA or protein production, and the assembly of a PCR-based nanofactory for gene delivery. Liposomes are the most successful drug delivery scaffolds ever developed with more than fifteen liposome-based drugs approved. Liposomes have long been investigated in the field of gene therapy as delivery vehicles for nucleic acids to overcome the barriers encountered by these molecules in vivo. In particular, the field of RNA interference-based drugs is very promising, with the first marketed formulation in 2018, and several others on their way. However, despite their long history of investigation, most of the structural and functional properties of the liposome-based drug delivery systems are inferred from bulk measurements. Therefore, the level of heterogeneity regarding, e.g. encapsulation efficiency, lipid composition, remains largely unknown within liposome preparations, which complicates the development of new formulations with improved therapeutic efficiency. Another important limitation is the observed membrane leakiness and premature drug release upon administration. Controlling the bio-distribution of a therapeutic drug is essential to minimize toxic side effects and enhance the efficiency of the treatment. To tackle this problem, the creation of targeting delivery systems with stimuli-responsive ability can improve the biodistribution profile of a drug and allow its delivery on demand. This work contributes to the convergence of the fields of synthetic biology, single-liposome biophysics and biomedicine, presenting different possible applications of vesicle bioreactors for the improvement of current RNA-based gene delivery systems.@en

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.@en

Natural life is extraordinarily complex, which by definition means that it has many interconnected and functioning parts. The goal of synthetic biology is to engineer living systems, though due to their very complexity they remain recalcitrant to engineering. What if it were possible to reduce the complexity to a finite amount of parts that are well understood and therefore possible to manipulate. That is the motivation for constructing a so called minimal cell.@en

The Christophe Danelon lab is involved in the long-term effort to construct an autonomous minimal cell using a bottom-up approach. Our goal is to achieve self-maintenance, selfreproduction, and evolution of a liposome compartment containing a minimal genome and a cell-free gene expression system. Self-reproduction requires splitting of the mother compartment into two daughter cells. The project described in this dissertation is part of the group’s attempts to create a minimal division unit for the synthetic cell. The development of a gene-driven, controllable, content-preserving liposome division strategy is an ongoing challenging task. Here, we reconstituted some of the organizational mechanisms for division of Escherichia coli in a cell-free system. In E. coli, cytokinesis is mediated by a multiprotein complex that forms a contractile ring-like structure at the division site. The ring is composed of the cytosolic filament-forming protein FtsZ, as well as its membrane anchoring proteins FtsA and ZipA. The Min system assists in the ring localization at mid-cell by oscillating from pole to pole. Using liposomes as a synthetic compartment and PURE system for cell-free gene expression, we reconstituted membrane-bound cytoskeletal structures and oscillating gradients of Min proteins for liposome constriction and dynamic organization of FtsZ filaments.@en

The creation of artificial cells with the minimal set of components to exhibit self-maintenance, self-reproducibility and evolvability (in other words, to be considered alive) is one of the most exciting areas within the field of synthetic biology. Such entities, here called minimal cells, are constructed by either the top-down or bottom-up approach. The top-down approach attempts to realize a minimal cell starting from an already existing unicellular organism and stripping down non-essential genes. In the bottom-up approach, separate biochemicals, such as phospholipids, DNA and proteins are assembled from scratch to reconstitute cell-like functions. On the way to tackle this curiosity-driven building challenge, we also expect to learn more about the most fundamental processes that define a living cell.@en

Humanity has achieved to decipher the most fundamental mechanics of cellular life. Nevertheless, despite intense efforts there are still considerable gaps in our understanding of cellular processes. Traditionally, biologists investigate life by observation of existing lifeforms. In order to assign functions to biological components, it is common practice to remove components from the system and then note the effect this has on the organism. Done repeatedly, this top-down approach allows for the creation of lifeforms with a reduced complexity, which makes it easier to fully map and model their cellular processes. In contrast, more and more additional effort is now exerted by the scientific community to recombine in vitro biological components in order to form a cellular lifeform. This bottom-up approach might not only yield in the end a minimal cellular system created de novo, but will further challenge us to verify and sophisticate our knowledge about cells. Gene expression through transcription and translation is the probably most fundamental process present in all cells existing today and any attempt of designing a minimal cellular system mimicking a real cell faithfully will have to involve these processes at its core. Thus, cell-free gene expression constitutes a key tool for the creation of a minimal cell. In this thesis we applied the bottom-up approach to investigate eukaryotic and prokaryotic microtubules, as well as the yeast ESCRT-III (endosomal sorting complex required for transport) and archaeal Cdv (cell division) system regarding their potential for cell-free expression and synthetic cell research. Overall, all proteins utilized in this thesis for cell-free expression (Mal3, BtubA, BtubB, BtubC, Vps20ΔC, Snf7, Vps2, Vps24, Vps4, CdvA, CdvB, and CdvC) have been synthesized by the PURE system at full-length. Therefore, expression itself was not a problem for any of the applied systems and the most critical step for each protein system was to evaluate if the expressed proteins were active regarding their functions and interactions. A key factor for each project was thus to find reliable testing conditions for the respective protein activity. For cell-free protein synthesis, we applied the commercially available PURE system, which is comprised exclusively of reconstituted components. A current drawback this system suffers from is that expression stops after a few hours due to unknown causes. This time interval is too short to reconstitute certain cellular functions and in the long run the design of a minimal cell will require a translation system that is more stable over time. Therefore, we attempted to enhance the expression lifetime of the PURE system by implementation of a semi-open system. However, no changes in duration of expression or yield was observed (Chapter 2). This result supports the hypothesis that neither accumulation of toxic waste products, nor the depletion of NTPs or amino acids are primarily responsible for break-down of PURE system activity over time. Another question we investigated was if it would be possible to regulate eukaryotic microtubules by expression of microtubule associated proteins (MAPs). We chose to attempt expression of the end-binding MAP Mal3 due to its ability to be expressed functionally in E. coli and its crucial role in organizing protein recruitment at the plus-end of microtubules. To visually confirm activity of expressed Mal3, we added it to microtubules together with the purified proteins Tea2 and Tip1, which are recruited by Mal3 to the microtubule plus-end (Chapter 3). In this plus-end tracking assay distinctive prove of the activity of expressed Mal3 was visually given by formation of comets at microtubule tips. A restriction faced with eukaryotic tubulin is that it cannot be synthesized by any prokaryotic expression system such as the PURE system. However, the tubulin homologues BtubA and BtubB have been previously discovered in bacteria of the genus Prosthecobacter, in which they form filaments similar to microtubules. We synthesized BtubA/B with the PURE system and were able to show that it was expressed at full-length and was fully capable of forming dynamic bacterial microtubules (Chapter 4). Assembly took mostly place on top of a supported lipid bilayer (SLB) to which the filaments were binding without addition of any cofactors. A fraction of labelled bacterial tubulin, which would not result in any filaments on its own, was added for visualization. Further, the capability of synthesized BtubC to recruit bacterial microtubules to lipid membranes beyond the tendency for binding already observed could be confirmed by flotation assays. Moreover, when expressed inside liposomes BtubA and BtubB formed filaments that were deforming the vesicles similar to what is known of encapsulated tubulin or actin. The encapsulated filaments could be disintegrated by intense laser illumination upon which vesicles appeared to reverse into their former shape. Overall, bacterial microtubules have the potential to become a useful tool for engineering synthetic cells under the premise that more proteins associated their regulation and function will be discovered. One of the challenges for the creation of a minimal cell is to achieve cell division and we explored the yeast ESCRT-III system in respect to its potential to facilitate division in a minimal cell setup (Chapter 5). However, assessing the activity of the four ESCRT-III proteins turned out to be difficult because of a lack of purified proteins. Nevertheless, we could assert membrane binding capabilities of the ESCRT proteins by flotation assays and colocalization to SLB membranes. The formation of filament complexes composed of expressed Vps20ΔC and Snf7 was confirmed by transmission electron microscopy. However, it is not certain if these structures are truly resembling ESCRT filaments. Membrane deformation initiated by expressed ESCRT-III proteins could not be achieved, which is in line with more recent literature that proved the dependency of the ESCRT complex on the ATPase Vps4 for this function. Vps4 can be expressed by the PURE system, but its ATPase activity was not analyzed, as consumption of ATP cannot be reliably detected in the PURE system and depolymerization of filaments would require more efficient visualization of filaments or filament complexes. Activity of expressed Cdv proteins could not be confirmed or analyzed (Chapter 6). It could only be determined that expressed CdvA, which is responsible for anchoring the Cdv complex to the membrane in archaea, did not bind to the lipid membranes we used in our settings. A possible reason for this could be differences in membrane composition between bacteria and archaea. As elaborated in Chapter 1 and 7, the terms and definitions that entail synthetics cells and the phenomenon of life are generally not very concise and rather arbitrary. We proposed that in most scientific work the respective definitions should be orientated with respect to the aim of the research and explicitly be restricted to the applied framework. Regarding a more general definition of lifeforms, we suggested that life is characterized by self-reproduction with variations, based on an internal information carrier. This definition excludes no lifeforms in general, but certain representatives of living entities which are uncapable of reproduction. Therefore, an even more basic and fundamental principle was proposed, according to which any kind of pattern which is capable of evolving could be considered alive. This definition not only includes all organisms generally considered alive but as well several other phenomena.@en