Towards the assembly of a minimal oscillator

Life must have started with lower degree of complexity and connectivity. This statement readily triggers the question how simple is the simplest representation of life? In different words and considering a constructive approach, what are the requirements for creating a minimal cell? This thesis sets the first step stones towards an ambitious goal of understanding fundamental principles of a minimal/synthetic life. The above-mentioned question has been addressed by two distinct but complementary investigation routes. In the top-down approach, in vivo reduction of genes of a living organism would reveal the minimal genome size that is essential to support life. Alternatively, in a bottom-up approach, essential cellular functions are constructed utilizing a minimal number of purified constituents for DNA, RNA and protein syntheses. Advances in such a protein-based in vitro minimal cell project has been owed to the recent development of the PURE system (Protein synthesis Using Recombinant Elements). The expression of genes into proteins is a fundamental and ubiquitous cellular process. The transfer of the DNA sequence information to proteins occurs in two steps forming the central dogma in molecular biology: the transcription of the gene in RNA and the subsequent translation of that RNA into a protein. During transcription the double-stranded DNA is copied by an RNA polymerase protein into a single stranded messenger RNA (mRNA). Translation is a more complex process involving many different components, with the ribosome as a key player. The synthesis of proteins from DNA templates inside lipid vesicles offers an elegant and unavoidable alternative to the exclusive use of purified constituents. By regulating the timely synthesis of the multiple proteins, the gene expression machinery is arguably at the heart of a programmable, genetically controlled, artificial cell. For a bimolecular assembly to be considered alive three major characteristics are inevitable: self-maintenance, self-replication and evolvability in response to the environmental signals. A distinguishing feature of a living cellular system, even in its simplest representation, is the compartment: a continuous membrane that acts as a functional interface to regulate the ionic and molecular exchanges with the environment. In Chapter 2, we established a porous matrix-assisted protocol to produce large and giant liposomes (micrometer size range) encapsulating the PURE system and DNA encoding a fluorescent reporter. The protocol describes immobilization of the liposomes on a glass coverslip and the methods to trigger internal gene expression and visualize those femtoliter-sized reactors using a laser scanning confocal microscope. We used several lipid compositions to better understand the microreactor properties. The results showed that gene expression is negatively influenced by the length of the phospholipid carbon chains. We believe that the main driving force for allocation of nutrients and tRNA across the membrane is the osmotic stress generated when diluting the liposomes in the feeding solution upon immobilization. Heterogeneity in the expression levels between individual vesicles is high and no obvious size dependency is observed. We developed a dual gene assay which proves that under this experimental condition, DNA concentration is not a limiting factor. Such a system provides a unique possibility to analyze compartmentalized gene expression inside individual vesicles over long time. To be able to rationally relate the measured protein expression signal to the encapsulation of multiple DNAs inside liposomes, in Chapter 3, we established the link between phenotype and genotype. The amounts of entrapped DNA molecules (the genotype) and synthesized proteins (the phenotype) were visualized by fluorescence confocal microscopy in single liposomes. We first showed that there exists no DNA crowding effect in giant liposomes. Our results suggest a compositional reorganization of DNA molecules within the first 30 min of the incubation, which leads to a large vesicle-to-vesicle heterogeneity. Moreover, we report no linear correlation between the amount of encapsulated genes and the level of output proteins. We then developed an assay based on expression of two orthogonally detectable fluorescent reporter proteins from two different DNA templates. Basically, the probabilities of single-color and double-color vesicles for three DNA concentrations were analyzed and compared to the expected values for Poisson statistics using the bulk DNA concentrations. The deviation of measured and Poisson probabilities was minimized when only 10 % of the bulk DNA was considered to be transcriptionally active. These findings will allow us to operate the expression of multiple genes in a controlled manner and to generate the output proteins with predictable dynamics in liposomes. An essential feature of a molecular assembly to be considered alive is self-reproduction, which implies that both the compartment and the genetic material supporting life self-replicate in a coordinated manner. A possible minimal divisome machinery in the absence of a cell wall would consist of FtsZ protofilaments anchored to the liposomes membrane by actin homologues, such as FtsA or ZipA. In Chapter 4, we investigated how membrane deforming proteins, such as NBAR, FtsZ and FtsA, could be integrated into the construction of a programmable minimal cell relying on gene expression inside liposomes. The aforementioned proteins were expressed in vitro from their corresponding genes. We then assessed liposome membrane sculpting activity of in situ expressed N-BAR protein in PURE system. Our results showed that the N-BAR domain protein could assemble onto the outer surface of liposomes and form tubular structures out of the membrane. Besides, we performed preliminary experiments on the in situ synthesis of full length FtsZ and FtsA from their corresponding gene. Oscillatory behaviors are fundamental in living organisms to regulate various functions in time and space. In Chapter 5, we aimed at utilizing our cell-free expression system as a platform for incorporation of regulatory networks towards achieving spatial and temporal dynamics of protein production. To be able to control the system's dynamics, the mRNA and protein lifetime has to be tunable. We achieved controlled degradation by supplementing the PURE system with purified MazF and ClpXP, which provides an additional level of control for mRNA and protein levels, respectively. We addressed the influence of the degradation machineries on transcription and translation. We then designed and characterized a number of genetic regulatory modules (two-stage cascade, repression, negative feedback) that will in the future be assembled into functional networks. The results presented in this thesis demonstrated that encapsulation of PURE system constituents in liposomes can be used as a primary experimental platform that allows us to implement various molecular machineries and regulatory circuits to recapitulate essential cell-like functions