AMOLF, The Netherlands
Reconstitution of biological processes from well-defined, individual components has enabled researchers to study increasing complex biological systems at the molecular and mechanistic level. To mimic cell or organelle membranes in these minimal systems, giant unilamellar vesicles (GUVs) are often used as simple models for membrane-bound compartments. As researchers aim to build a minimal synthetic cell from the bottom up, reconstitution of cellular processes in GUVs will be particularly valuable to include transport processes across membranes and a mechanically responsive compartment. However, reliable GUV fabrication remains a major experimental bottle-neck, in particular if they are to encapsulate functional biomolecules. Here, we will present our recent work on optimising a double-emulsion method for producing functionalised GUVs called continuous droplet interface crossing encapsulation (cDICE). By optimising the protocol and tightly controlling experimental conditions, we improved the efficiency, ease, and, most importantly, reproducibility of cDICE-based GUV formation. We show cDICE can produce GUVs containing a multitude of minimal systems, including a minimal actin cytoskeleton and bacterial chromosomes. By allowing the formation of bespoke liposomes with good yields and reproducibility, optimised cDICE promises to become a standard method for making functionalised vesicles for the biophysics and synthetic biology communities.
Delft University of Technology, The Netherlands
All life is made of cells, which are the smallest units of life that are themselves alive. Cells in turn are made of a complex mixture of molecules that are themselves lifeless. How do lifeless molecules interact to form a living cell that sustains itself, grows and replicates? In the large nation-wide BaSyC program, we aim to build a synthetic cell from the bottom up in order to understand ‘how life works’. Our own research focused on the challenge of reconstituting a minimal cell division apparatus based on the mammalian cytoskeletal protein actin. The animal cell division apparatus is mechanically the simplest, since animal cells lack a cell wall and an actin-based ring is able to constrict the membrane. In this talk I will discuss our efforts to reconstitute giant unilamellar vesicles (GUVs) with an active actin cortex that can drive membrane deformation. We show that large quantities of defect-free cell-sized GUVs containing actin in physiological buffer can be generated by an optimized emulsion method that we term emulsion-droplet interface crossing encapsulation (eDICE). Using eDICE, we efficiently encapsulate actin with regulatory proteins such as nucleators, with control over membrane anchoring. We show that dendritic actin cortices are sufficient to stiffen the membrane and to produce and trap globally deformed membrane shapes, which strikingly persist orders of magnitude longer than the actin turnover times. We further demonstrate that such cortices are capable of producing filopodia-like membrane protrusions. The next step is to include myosin motors to drive membrane invagination.
Wageningen University and Research, The Netherlands
The emerging field of synthetic biology is continuously exploring new avenues and techniques to shed light on how living systems work, and to synthesize or altogether re-design biological systems. In this talk, I will show you that microfluidic technology is a powerful tool to create and manipulate synthetic cell containers. Recent years have seen fantastic utilization of lab-on-a-chip set ups to form three-dimensional biomimicking confinements in a controlled and high-throughput fashion. A common theme towards making these containers seems to be a process akin to bubble-blowing, which can be used to make single emulsions (water-in-oil droplets), double emulsions (water-in-oil-in-water droplets), and liposomes (GUVs), with increasing levels of production complexity and cell-mimicking capabilities. In particular, I will discuss octanol-assisted liposome assembly (OLA) technique to produce cell-sized, monodispersed, unilamellar liposomes with an excellent encapsulation efficiency. After briefly touching upon various things achieved using OLA such growth and division of liposomes and drug-screening assays, I will focus on compartmentalization of OLA-based liposomes using biomolecular condensates, formed through the process of liquid-liquid phase separation. I will also talk about the interactions of these condensates with the liposomal membrane, a topic of increasing importance. In conclusion, such bubble-blowing micromachines are valuable tools for creating synthetic cells.