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.

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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.

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.