1: Leibniz-Institut für Polymerforschung Dresden e.V., Germany; 2: Centro de Investigaciones Biológicas Margarita Salas (CIB-CSIC), Spain
Polymer microgels with their porous nature serve as an open reactor where proteins, mRNA and other (macro-)molecules can freely diffuse in and out. On the other hand, the polymer network provides a certain density compared to a solution and offers the possibility to couple DNA or proteins for a spatial control of processes. Here, we show how polymer microgels made from hyaluronic acid can be produced by droplet microfluidics with desired size, shape and porosity, and how they can be used for different applications in synthetic biology.
In a first application, linear DNA that encodes a certain protein was coupled to polymer microgels to perform cell-free protein synthesis (CFPS). By incorporating nitrilotriacetic acid groups in the polymer microgels the synthesized proteins carrying a His-Tag can be directly immobilized after CFPS. So far, we studied the process by using the model green fluorescent protein and applied it for filamenting temperature-sensitive mutant (FtsZ) protein.
In another application, we used polymer microgels as nucleoid mimic in bacterial cell division. One of the investigated proteins, SmlA, specifically binds to a short DNA sequence, named SBScons. This DNA was covalently coupled to the polymer microgels to mimic the DNA region of the cell. Afterwards, the interaction of SmlA, FtsZ and ribosomes with the polymer microgels as well as the influence of porosity on the diffusion of the fluorescently labeled proteins were studied by confocal laser scanning microscopy.
With our investigations, we would like to show the diversity of hyaluronic acid-based polymer microgels and how their applications could give important impacts in the field of synthetic biology.
Johns Hopkins University, United States of America
Living organisms detect and produce gradients for a variety of different tasks, including morphogenesis, chemotaxis, and distributed computation. Here, we demonstrate an automated method for photopatterning multi-domain hydrogels, and then apply that method towards building hydrogels capable of RNA gradient generation. The automated photopatterning method flows photosensitive hydrogel ‘inks’ into a microfluidic flow cell and can then be patterned into a variety of shapes and sizes within 10 – 500 µm using maskless photolithography. To build an RNA gradient generator, we anchor transcription templates within hydrogel posts that act as chemical sources, constantly producing RNA via RNA Polymerase-mediated transcription. The RNA diffuses out of the hydrogel and is degraded by RNAse A/T1. Control over the hydrogel positions, anchored transcription template concentrations and enzyme RNA production and degradation rates enables the construction of sets of RNA gradients tunable magnitudes and widths. To visualize these gradients, we use “reporter” hydrogels containing DNA complexes that react with the RNA to produce a fluorescent signal. Multiple photopatterned source hydrogels could also be used to form more complex additive gradients, more closely recreating environments in cell populations. This platform also makes it possible to direct local self-assembly of nucleic acid responsive nanostructures and nanoparticles. We envision building a distributed computation system by creating layers of interconnected sender-receiver pairs (using activatable templates) to perform various computations such as spatial shape recognition.
MeBioS, KU Leuven, Belgium
Giant unilamellar vesicles (GUVs) provide a scaffold for artificial cell construction. Researcher have been successful in incorporating a wide variety of (membrane)proteins, expression constructs, molecules… in these cell like vesicles. However the incorporation of transmembrane proteins remains laborious and typically requires detergents that disturb protein conformation/function. Hence, to expand the applicability of artificial cells, a detergentless, simple and robust method for membrane protein incorporation into GUVs would be desirable.
One of the most attractive strategies to do so is by using styrene maleic acid lipid particles (SMALPs). SMA enables the easy solubilization and purification of transmembrane proteins into lipid nanodiscs. Since no detergents are required for this solubilization, the transmembrane proteins remain in their native lipid environment which maximizes the likelihood of retaining their function upon their subsequent incorporation into GUVs.
In our research group we are bridging the gap between SMALP technology for protein purification and GUV formation technologies. By engineering the construction of GUVs within droplet-based microfluidic systems, SMALPs can be destabilized at a droplet interface and fused into a GUV. Several sets of SMALPs can be combined in a single droplet, creating an artificial cell-like membrane with a well-defined transmembrane protein content. This microfluidic droplet-based method has the potential to drive the generation of artificial cells with increased responsivity to environmental cues.
VU Amsterdam, the Netherlands
The mechanical properties of the cell are dictated by the cytoskeleton, which consists of an interlinked network of filaments. The cytoskeleton not only determines the cell shape but also drives vital processes such as cell motility and division. Successful reconstitution of the cytoskeleton inside a liposome is thus a crucial step for creating a viable artificial cell. It would therefore be desirable to have a robust and fast technique at one’s disposal to characterize the biomechanics of the cytoskeleton both in artificial and living cells. To this end, we demonstrate here quantitative acoustophoresis: a rather simple but reliable and precise method to quantify the biomechanics of single bioparticles.
In acoustophoresis, a standing acoustic wave is generated inside a flow channel, and the velocities of bioparticles are measured as they move towards the resulting field node. These velocities depend on the acoustic contrast factor, a function of both samples’ density and compressibility. Since our approach allows us to measure the density independently by different means, we are thus not only able to measure particle’s compressibility but also follow its change over time.
Here, we first showcase the resolution capabilities of our instrument using um-sized polymer microspheres in proof-of-concept experiments. We next applied our assay to characterize the mechanics of artificial liposomes (some containing a 3D gel network as cytoskeleton mimic) and various cell lines. These results demonstrate that our method provides a promising tool to measure changes in cellular compressibility, either drug-induced or occurring naturally during the cell cycle.