Creating synthetic eukaryotic cells with giant lipid vesicles and microfluidics
Giant unilamellar vesicles (GUVs) are used extensively in bottom-up synthetic biology as a scaffold to construct synthetic cells de novo. Microfluidic-based approaches for GUV production show great potential for encapsulating large biomolecules required for mimicking life-like functions (Yandrapalli et al. Micromachines, 2020; Love et al. Angew Chemie, 2020). First, I will briefly show how the combination of microfluidics and GUVs can provide high-throughput membrane analysis (Robinson, Adv. Biosyst. 2019; Yandrapalli and Robinson, Lab Chip, 2019), which we have used for permeation measurements (Bhatia, Soft Matter, 2020), applying external forces to individual vesicles (Robinson et al. Lab on a Chip 2014; Sturzenegger et al. Soft Matter 2016; Robinson et al. ChemBioChem 2019), and for membrane fusion studies (Lira, Robinson et al. Biophys. J. 2019). This talk will focus on our latest results of how microfluidics can aid in bottom-up synthetic biology.
The first is a microfluidic design able to produce surfactant-free pure lipid GUVs in a high-throughput and monodisperse manner (Yandrapalli et al. bioRxiv, 2020, doi:10.1101/2020.10.23.346932). The major advancement is that the lipid-containing carrier oil is biocompatible and the vesicles are produced in the absence of block co-polymers or surfactants that can affect their biocompatibility. The design can produce homogenously sized GUVs with tuneable diameters from 10 to 120 µm. Encapsulation is uniform and we show that the membranes are leakage-free and oil-free by measuring the diffusion of lipids via FRAP measurements. Next, we present an advanced version of this device able encapsulate two sub-populations of nanometre-sized vesicles (LUVs) for the purpose of establishing multiple enzymatic reactions across membrane-bound compartments, therefore mimicking eukaryotic cells. The final synthetic cell comprises three compartments, and couples three separate enzymes for cascade reactions. Our microfluidic technique provides a high degree of control over the intra-vesicular conditions such as enzyme concentrations, buffers, and the number of inner compartments - essential for bottom-up assembly of artificial eukaryotic cells. This work demonstrates the effectiveness of microfluidics for the bottom-up construction, handling and analysis of artificial cell constructs.
Bhatia, T., Robinson, T., and Dimova, R. (2020). Membrane permeability to water measured by microfluidic trapping of giant vesicles. Soft Matter 16, 7359–7369.
Lira, R. B., Robinson, T., Dimova, R., and Riske, K. A. (2018). Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study. Biophys. J. 116, 79–91.
Love, C., Steinkühler, J., Gonzales, D. T., Yandrapalli, N., Robinson, T., Dimova, R., and Tang, T.-Y. D. (2020). Reversible pH‐Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions. Angew. Chemie Int. Ed. 59, 5950–5957.
Robinson, T. (2019). Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review. Adv. Biosyst., 1800318.
Robinson, T., Verboket, P. E., Eyer, K., and Dittrich, P. S. (2014). Controllable electrofusion of lipid vesicles: initiation and analysis of reactions within biomimetic containers. Lab Chip, 2852–2859.
Robinson, T., and Dittrich, P. S. (2019). Observations of membrane domain reorganization in mechanically compressed artificial cells. ChemBioChem, cbic.201900167.
Sturzenegger, F., Robinson, T., Hess, D., and Dittrich, P. S. (2016). Membranes under shear stress: visualization of non-equilibrium domain patterns and domain fusion in a microfluidic device. Soft Matter 12, 5072–5076.
Yandrapalli, N., and Robinson, T. (2019). Ultra-high capacity microfluidic trapping of giant vesicles for high-throughput membrane studies. Lab Chip 19, 626–633.
Yandrapalli, N., Seemann, T., and Robinson, T. (2020). On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis. Micromachines 11, 285.
Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.