Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

Cell-Free Expressed Membraneless Organelles Inhibit Translation in Synthetic Cells.

Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.

Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

Cell-free expressed membraneless organelles sequester RNA in synthetic cells.

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

Toward synthetic life: Biomimetic synthetic cell communication.

Engineering synthetic minimal cells provide a controllable chassis for studying the biochemical principles of natural life, increasing our understanding of complex biological processes. Recently, synthetic cell engineering has enabled communication between both natural live cells and other synthetic cells. A system such as these enable studying interactions between populations of cells, both natural and artificial, and engineering small molecule cell communication protocols for a variety of basic research and practical applications. In this review, we summarize recent progress in engineering communication between synthetic and natural cells, and we speculate about the possible future directions of this work.

Place learning overrides innate behaviors in Drosophila.

Animals in a natural environment confront many sensory cues. Some of these cues bias behavioral decisions independent of experience, and action selection can reveal a stimulus-response (S-R) connection. However, in a changing environment it would be a benefit for an animal to update behavioral action selection based on experience, and learning might modify even strong S-R relationships. How animals use learning to modify S-R relationships is a largely open question. Three sensory stimuli, air, light, and gravity sources were presented to individual Drosophila melanogaster in both naïve and place conditioning situations. Flies were tested for a potential modification of the S-R relationships of anemotaxis, phototaxis, and negative gravitaxis by a contingency that associated place with high temperature. With two stimuli, significant S-R relationships were abandoned when the cue was in conflict with the place learning contingency. The role of the dunce (dnc) cAMP-phosphodiesterase and the rutabaga (rut) adenylyl cyclase were examined in all conditions. Both dnc1 and rut2080 mutant flies failed to display significant S-R relationships with two attractive cues, and have characteristically lower conditioning scores under most conditions. Thus, learning can have profound effects on separate native S-R relationships in multiple contexts, and mutation of the dnc and rut genes reveal complex effects on behavior.