Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells

<p>The bottom-up assembly of multi-compartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform which is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane (PDMS) channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments <i>via </i>the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentation effects on biochemical reactions but is also applicable for developing advanced drug-delivery systems as well as minimal cells in the field of bottom-up synthetic biology.</p>

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells

<p>The bottom-up assembly of multi-compartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform which is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane (PDMS) channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments <i>via </i>the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentation effects on biochemical reactions but is also applicable for developing advanced drug-delivery systems as well as minimal cells in the field of bottom-up synthetic biology.</p>

Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.

Genetic requirements for cell division in a genomically minimal cell

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics resulting in irregular morphologies. The addition of 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents significantly less morphological variation than JCVI-syn3.0. We further identified seven of these 19 genes, including two known cell division genes ftsZ and sepF and five genes of unknown function, required together to restore cell morphology and division similar to JCVI-syn1.0. This surprising result emphasizes the polygenic nature of cell morphology, as well as the importance of a Z-ring and membrane properties in the physiology of genomically minimal cells.

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

Executing gene circuits by cell-free transcription−translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.