Coacervate microdroplets
A protocell was collected from coacervate droplets and bacteria
Elena Kleshchenko, PCR.news
The ambitious goal of synthetic biology is the construction of artificial cells "from the bottom up", from the simplest components. There are such projects in the Netherlands, USA, The European Union. But in order to replace the inert capsules in which the enzymes are immobilized with living or similar living cells, it is necessary not only to "assemble" the enzymatic chains and the replicative apparatus, but also to construct the structural components of the cell and provide a connection between them.
Researchers from the UK, China and France solved the structural problem by placing fragments of destroyed bacterial cells in a synthetic framework. They called this framework "coacervate microcapsules". Russian science lovers are familiar with this term according to A.I. Oparin's hypothesis about biogenesis: coacervates are membrane—free droplets of a dense liquid phase that form and spontaneously separate from aqueous solutions. The authors of the new work coacervates contained a synthetic polymer poly(diallyldimethylammonium chloride) (PDDA) and ATP.
Drops with a diameter of 5-30 microns were used to capture two types of bacteria: Escherichia coli were localized inside the drops, Pseudomonas aeruginosa Pseudomonas aeruginosa (strain PAO1) — on the surface. The bacteria were then lysed using cell wall hydrolase (lysozyme) and an antimicrobial peptide (melittin). The result was artificial cells — coacervate droplets covered with a PAO1 membrane and containing active enzymes inside, protein synthesis mechanisms, water-filled vacuoles also clad with a PAO1 membrane, and bacterial DNA genomes (plasmids). Protein typing by liquid chromatography—mass spectrometry showed that the protocells retained 2,237 bacterial proteins, or approximately 83% of the combined library.
The authors demonstrated that the protocell is capable of transcription and translation, that is, producing small amounts of protein, as well as generating ATP due to glycolysis.
In natural cells, in addition to membrane organelles, there are membrane-free components that have arisen due to the liquid-liquid phase separation. They are involved in RNA metabolism, ribosome biogenesis, DNA damage response, and signal transmission. The authors confirmed that subcellular organization can also be achieved in the coacervate protocell. With the help of enzymes, they split bacterial DNA into short strands in the "cytoplasm" of their artificial cells, then added a negatively charged polymer (carboxymethyldextran) and histones — proteins with which DNA is bound in the nucleus. Fragments with DNA histones condensed into a nucleus-like structure. Perhaps, in living cells, DNA is compartmentalized in a similar way. Of course, the nucleus in artificial cells is non-functional, but the authors suggest that functions can be acquired in the future.
The problem of dividing and changing the shape of an artificial cell has not been solved. Many research groups are working on it using cytoskeleton proteins, which give shape to natural cells, are responsible for their movement and division. It is difficult to change the shape of coacervates in this way, but the authors have taken a step in this direction. Actin, one of the main proteins of the cytoskeleton, polymerized and formed networks in the "cytoplasm" of artificial cells, but they remained rounded. Interestingly, when the authors placed live bacteria inside the coacervates, the artificial cells became asymmetric, similar to amoebas.
A drawing from the press release of the University of Bristol Pioneering research using bacteria brings scientists a step closer to creating artificial cells with lifelike functionality – VM.
The authors implanted live E.coli cells into their designs to provide energy. The bacteria performed the role of mitochondria — they produced ATP and secreted it externally, into the "cytoplasm". (As is known, the mitochondria of modern cells originated from symbiotic alpha-proteobacteria.) Usually, an artificial cell captured from 10 to 50 bacterial cells. Many of them remained viable after capture for 3 hours. ATP production was stimulated by the addition of glucose. This solution prolonged the enzymatic activity. gene expression and increased the level of actin polymerization in the structure
Such an approach cannot yet be called the construction of a cell "from the floor level": bacterial cells used as building materials are complex and insufficiently understood in themselves. The genome of the "minimal cell" that Craig Venter's staff received in 2016 still contained about a third of the genes whose functions were unknown and nevertheless necessary. To build a fully controllable artificial cell, it is necessary to understand how all its components interact with each other. Nevertheless, the new work demonstrates an additional "degree of freedom" for synthetic biology.
Previously, attempts have been made to create artificial cells based on living cells or cellular components, but this is the first time such a radical approach has been used. The authors suggest that the coacervate platform will allow combining the most diverse functions of bacterial cells. For example, implantation of living photosynthetic or sulfate-reducing bacteria into the protocells will help to create bionic systems that can exist due to light or chemosynthesis in anaerobic conditions. One can also imagine a protocellular system where bacterial bioluminescence provides photosynthetic bacteria with light energy, and those in turn supply carbohydrates and oxygen to the "mitochondria" from E. coli cells.
This study once again makes you think about what life is, he notes Amy Evdal from the University of Nijmegen (Netherlands). Scientists define living beings as having certain characteristics: cellular and subcellular compartmentalization, metabolism, the ability to store and process information, regeneration, etc. There are artificial cells that reproduce some of these traits. But if life is an emergent property (one that appears in the system as a whole and is not inherent in its individual elements), then an artificial cell will not become alive until it combines all the necessary properties of a living one. Perhaps a coacervate platform
Article by Xu et al. The living material assembly of bacteriogenic protocells is published in the journal Nature.
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