Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB, which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by a light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.

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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

DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (protein synthesis using recombinant elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, there is an urgent need to improve the system's performance and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors (IFs) by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, the depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNA-hydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

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