pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

The peptidoglycan cell wall is a defining structural feature of the bacterial kingdom. Curiously, some bacteria have the ability to switch to a wall-free or ‘L-form’ state. Although known for decades, the general properties of L-forms are poorly understood, largely due to the lack of systematic analysis of L-forms in the molecular biology era. Here we show that inhibition of peptidoglycan precursor synthesis promotes the generation of L-forms from both Gram-positive and Gram-negative bacteria. We show that the L-forms generated have in common a mechanism of proliferation involving membrane blebbing and tubulation, which is dependent on an altered rate of membrane synthesis. Crucially, this mode of proliferation is independent of the essential FtsZ based division machinery. Our results suggest that the L-form mode of proliferation is conserved across the bacterial kingdom, reinforcing the idea that it could have been used in primitive cells, and opening up its use in the generation of synthetic cells.

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Actuation of synthetic cells with proton gradients generated by light-harvesting E. coli

10.21203/rs.3.rs-82114/v1 ◽

2020 ◽

Author(s):

Kevin Jahnke ◽

Noah Ritzmann ◽

Julius Fichtler ◽

Anna Nitschke ◽

Yannik Dreher ◽

...

Keyword(s):

Lipid Vesicles ◽

Cellular Systems ◽

Top Down ◽

Ph Gradients ◽

Proton Gradients ◽

Bottom Up ◽

E Coli ◽

Genetically Engineer ◽

Synthetic Cells ◽

Dna Triplex

Abstract Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness new types of living systems. Both approaches, however, face their own challenges towards biotechnological and biomedical applications. Here, we realize a strategic merger 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 as organelle mimics in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we can 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. By attaching a polymerized DNA origami plate to the DNA triplex motif, we obtain a cytoskeleton mimic that considerably deforms lipid vesicles in a pH-responsive manner. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells as potent microreactors.

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Mimicking Adhesion in Minimal Synthetic Cells

Advanced Biosystems ◽

10.1002/adbi.201800333 ◽

2019 ◽

Vol 3 (6) ◽

pp. 1800333 ◽

Cited By ~ 10

Author(s):

Solveig M. Bartelt ◽

Elizaveta Chervyachkova ◽

Julia Ricken ◽

Seraphine V. Wegner

Keyword(s):

Synthetic Cells

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Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

Nature Communications ◽

10.1038/s41467-020-20124-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Wiggert J. Altenburg ◽

N. Amy Yewdall ◽

Daan F. M. Vervoort ◽

Marleen H. M. E. van Stevendaal ◽

Alexander F. Mason ◽

...

Keyword(s):

Spatial Organization ◽

Nitrilotriacetic Acid ◽

Binding Motif ◽

Biologically Relevant ◽

Enzyme Cascade ◽

Synthetic Cell ◽

Synthetic Cells ◽

Membrane Bound ◽

High Concentrations ◽

High Level

AbstractThe cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

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