With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.

Holding tight: An artificial membrane nanopore assembled from DNA oligonucleotides carries porphyrin tags (red), which anchor the nanostructure into the lipid bilayer. The porphyrin moieties also act as fluorescent dyes to aid the microscopic visualization of the DNA nanopore.

DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance tenfold compared to previous man-made channels. In our design, nineteen cholesterol-tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.

The cytoskeleton is an essential component of a cell. It controls the cell shape, establishes the internal organization, and performs vital biological functions. Building synthetic cytoskeletons that mimic key features of their natural counterparts delineates a crucial step towards synthetic cells assembled from the bottom up. To this end, DNA nanotechnology represents one of the most promising routes, given the inherent sequence specificity, addressability and programmability of DNA. Here we demonstrate functional DNA-based cytoskeletons operating in microfluidic cell-sized compartments. The synthetic cytoskeletons consist of DNA tiles self-assembled into filament networks. These filaments can be rationally designed and controlled to imitate features of natural cytoskeletons, including reversible assembly and ATP-triggered polymerization, and we also explore their potential for guided vesicle transport in cell-sized confinement. Also, they possess engineerable characteristics, including assembly and disassembly powered by DNA hybridization or aptamer–target interactions and autonomous transport of gold nanoparticles. This work underpins DNA nanotechnology as a key player in building synthetic cells.

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multi-compartment synthetic cells. Cellular components are enclosed by giant unilamellar vesicles (GUVs), produced - at milliliter scale - directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Towards this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil-surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein alpha-hemolysin and assess the membrane quality with cryoTEM, FRAP and zeta-potential measurements as well as confocal fluorescent imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques - offering high volumes, a flexible choice of lipid compositions and buffer conditions, the straight-forward co-reconstitution of proteins and a high encapsulation efficiency of biomolecules and even large compounds including cells. We thereby provide a simple, robust and broadly applicable strategy to mass-produce complex multi-component GUVs for high-throughput testing in synthetic biology and biomedicine which can directly be implemented in laboratories around the world.

Due to their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway. Ions flow at the DNA-lipid interface as the lipid head groups tilt towards the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here, with the smallest possible DNA-based ion channel, offers a route to design a new class of synthetic ion channels with maximum simplicity.