Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System.

A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid-derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP-regenerating and oxygen-scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom-up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.

DNA Segregation in Natural and Synthetic Minimal Systems.

Faithful segregation of replicated genomes to dividing daughter cells is a major hallmark of cellular life and needs to be part of the future design of the robustly proliferating minimal cell. So far, the complexity of eukaryotic chromosome segregation machineries has limited their applicability to synthetic systems. Prokaryotic plasmid segregation machineries offer promising alternative tools for bottom-up synthetic biology, with the first three-component DNA segregation system being reconstituted a decade ago. In this review, the mechanisms underlying DNA segregation in prokaryotes, with a particular focus on segregation of plasmids and chromosomal replication origins are reviewed, along with a brief discussion of archaeal and eukaryotic systems. In addition, this review shows how in vitro reconstitution has allowed deeper insights into these processes and discusses possible applications of these machineries for a minimal synthetic segrosome as well as the challenge of its coupling to a minimal replisome.

Cytoskeletal and Actin-Based Polymerization Motors and Their Role in Minimal Cell Design.

Life implies motion. In cells, protein-based active molecular machines drive cell locomotion and intracellular transport, control cell shape, segregate genetic material, and split a cell in two parts. Key players among molecular machines driving these various cell functions are the cytoskeleton and motor proteins that convert chemical bound energy into mechanical work. Findings over the last decades in the field of in vitro reconstitutions of cytoskeletal and motor proteins have elucidated mechanistic details of these active protein systems. For example, a complex spatial and temporal interplay between the cytoskeleton and motor proteins is responsible for the translation of chemically bound energy into (directed) movement and force generation, which eventually governs the emergence of complex cellular functions. Understanding these mechanisms and the design principles of the cytoskeleton and motor proteins builds the basis for mimicking fundamental life processes. Here, a brief overview of actin, prokaryotic actin analogs, and motor proteins and their potential role in the design of a minimal cell from the bottom-up is provided.

Functional Modules of Minimal Cell Division for Synthetic Biology.

Cellular reproduction is one of the fundamental hallmarks of life. Therefore, the development of a minimal division machinery capable of proper genome condensation and organization, mid-cell positioning and segregation in space and time, and the final septation process constitute a fundamental challenge for synthetic biology. It is therefore important to be able to engineer such modules for the production of artificial minimal cells. A bottom-up assembly of molecular machines from bulk biochemicals complemented by in vivo experiments as well as computational modelling helps to approach such key cellular processes. Here, minimal functional modules involved in genome segregation and the division machinery and their spatial organization and positioning are reviewed, setting into perspective the design of a minimal cell. Furthermore, the milestones of recent in vitro reconstitution experiments in the context of cell division are discussed and their role in shedding light on fundamental cellular mechanisms that constitute spatiotemporal order is described. Lastly, current challenges in the field of bottom-up synthetic biology as well as possible future developments toward the development of minimal biomimetic systems are discussed.

Motility and chemotaxis of bacteria-driven microswimmers fabricated using antigen 43-mediated biotin display.

Bacteria-driven biohybrid microswimmers (bacteriabots) combine synthetic cargo with motile living bacteria that enable propulsion and steering. Although fabrication and potential use of such bacteriabots have attracted much attention, existing methods of fabrication require an extensive sample preparation that can drastically decrease the viability and motility of bacteria. Moreover, chemotactic behavior of bacteriabots in a liquid medium with chemical gradients has remained largely unclear. To overcome these shortcomings, we designed Escherichia coli to autonomously display biotin on its cell surface via the engineered autotransporter antigen 43 and thus to bind streptavidin-coated cargo. We show that the cargo attachment to these bacteria is greatly enhanced by motility and occurs predominantly at the cell poles, which is greatly beneficial for the fabrication of motile bacteriabots. We further performed a systemic study to understand and optimize the ability of these bacteriabots to follow chemical gradients. We demonstrate that the chemotaxis of bacteriabots is primarily limited by the cargo-dependent reduction of swimming speed and show that the fabrication of bacteriabots using elongated E. coli cells can be used to overcome this limitation.

The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism.

The pseudotetrasaccharide acarbose is a medically relevant secondary metabolite produced by strains of the genera Actinoplanes and Streptomyces. In this study gene products involved in acarbose metabolism were identified by analyzing the cytosolic and extracellular proteome of Actinoplanes sp. SE50/110 cultures grown in a high-maltose minimal medium. The analysis by 2D protein gel electrophoresis of cytosolic proteins of Actinoplanes sp. SE50/110 resulted in 318 protein spots and 162 identified proteins. Nine of those were acarbose cluster proteins (Acb-proteins), namely AcbB, AcbD, AcbE, AcbK, AcbL, AcbN, AcbR, AcbV and AcbZ. The analysis of proteins in the extracellular space of Actinoplanes sp. SE50/110 cultures resulted in about 100 protein spots and 22 identified proteins. The identifications included the three acarbose gene cluster proteins AcbD, AcbE and AcbZ. After their identification, proteins were classified into functional groups. The dominant functional groups were the carbohydrate binding, carbohydrate cleavage and carbohydrate transport proteins. The other functional groups included protein cleavage, amino acid degradation, nucleic acid cleavage and a number of functionally uncharacterized proteins. In addition, signal peptide structures of extracellularly found proteins were analyzed. Of the 22 detected proteins 19 contained signal peptides, while 2 had N-terminal transmembrane helices explaining their localization. The only protein having neither of them was enolase. Under the conditions applied, the secretome of Actinoplanes sp. SE50/110 was dominated by seven proteins involved in carbohydrate metabolism (PulA, AcbE, AcbD, MalE, AglE, CbpA and Cgt). Of special interest were the identified extracellular pullulanase PulA and the two solute-binding proteins MalE and AglE. The identifications suggest that Actinoplanes sp. SE50/110 has two maltose/maltodextrin import systems. We postulate the identified MalEFG transport system of Actinoplanes sp. SE50/100 as the missing acarbose-metabolite importer and present a model of acarbose metabolism that is extended by the newly identified gene products.