Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two‐photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water‐soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two‐photon printing inside the compartment does not destroy the GUVs. Beyond this proof‐of‐principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two‐photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology.
Balancing the maximal efficacy of interferon gamma (IFN-ɣ)-based therapies with its side effects is a great challenge for future cytokine treatments. To achieve this, the development of single-cell technologies that study IFN-ɣ release in correlation with antitumor activity would represent a huge step forward. To this end, droplet-based microfluidics is employed to quantitatively investigate IFN-ɣ secretion from single natural killer (NK) cells in correlation with their cytotoxic activity against a specific target. The method relies on co-encapsulation of NK-92 cells, target cancer cells, polystyrene beads conjugated with specific IFN-ɣ capture antibodies, and fluorescently labeled detection antibodies inside water-in-oil compartments. The secreted cytokines are captured and detected by localized fluorescence at the periphery of the beads. NK-92's cytotoxicity is evaluated simultaneously by means of a fluorescent DNA intercalating agent, which penetrates the membranes of dead target cells. To deepen the understanding of the role of the cytokine in antitumor immunomodulation, the impact of different doses of human recombinant IFN-ɣ on the cytolytic activity of NK-92 cells shows a trend that the higher the dose the lower the cytolytic activity of NK cells. The developed method represents a simple quantitative approach to unravel the complex heterogeneity of NK cells toward IFN-ɣ secretion and cytolytic activity.
Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.