Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer

Takahashi, Hajime; Ogawa, Atsushi

doi:10.1021/acssynbio.0c00173

Cited by 7 publications

(7 citation statements)

References 22 publications

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“…Within this strategy, various experimental systems, such as ATP and protein synthesis circuits in liposomes driven by light irradiation, [ 73 ] actin‐encapsulating liposomes exhibiting light‐responsive transformation, [ 74 ] and stochastic enzymatic reactions inside liposomes [ 75 ] have been constructed. However, because the maximum size of liposomes reported so far is ≈2 mm, [ 76 ] without any structural reinforcement for the strength of liposomes using hydrogels or other macromolecular network, [ 77 ] the larger liposomes are difficult to be realized. This size restriction could be a barrier for the implementation of a highly hierarchical structure inside a liposome.…”

Section: Liposome Assembling Technologies For Cellular Chemical Aimentioning

confidence: 99%

Molecular Cybernetics: Challenges toward Cellular Chemical Artificial Intelligence

Murata

Toyota

Nomura

et al. 2022

Adv Funct Materials

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Research on so-called "chemical artificial intelligence" (CAI) is an emerging field with the aim of constructing information-processing systems with learning capabilities based on chemical methodologies. This can be regarded as an attempt to reconstruct Cybernetics using molecular based systems. Many chemical reaction systems with computational abilities are proposed, but most are fixed functions that deliver molecular output for a given molecular input. On the other hand, chemical AI is a system with learning capability; namely, the output should be variable and gradually change upon repeated molecular inputs. In this paper, a compartmentalization approach for implementing cellular chemical AI using liposomes is discussed. The existing studies in terms of the methods used for assembling systems consisting of many liposomes with different functions, methods for achieving recursiveness and plasticity in chemical reaction systems, and methods for reconfiguring the network topology by liposome deformation are reviewed. Issues that must be addressed in order to realize chemical AI are also identified.

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Section: Liposome Assembling Technologies For Cellular Chemical Aimentioning

confidence: 99%

Molecular Cybernetics: Challenges toward Cellular Chemical Artificial Intelligence

Murata

Toyota

Nomura

et al. 2022

Adv Funct Materials

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“…Such GUVs are often prepared by the water-in-oil (W/O) emulsion method, 20 which produces GUVs with a wide range of size distributions. 21 Proteins can be encapsulated in these GUVs 22,23 or asymmetrically localized on the inner or outer leaflet. 20 GUVs are appropriate for mimicking animal cells but not for mimicking bacterial cells, which are mimicked by large unilamellar vesicles (LUVs), i.e., liposomes 100−1000 nm in diameter, similar in size to viruses, exosomes, and bacteria.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Such GUVs are often prepared by the water-in-oil (W/O) emulsion method, which produces GUVs with a wide range of size distributions . Proteins can be encapsulated in these GUVs , or asymmetrically localized on the inner or outer leaflet . GUVs are appropriate for mimicking animal cells but not for mimicking bacterial cells, which are mimicked by large unilamellar vesicles (LUVs), i.e., liposomes 100–1000 nm in diameter, similar in size to viruses, exosomes, and bacteria. ,, The surface of LUVs, which are usually prepared using an extruder (the extruder method), can be decorated by proteins utilizing a chemical reaction with membrane-inserted lipid molecules linked with a functional group.…”

Section: Introductionmentioning

confidence: 99%

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane

et al. 2023

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Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 μm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional waterin-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500−2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

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“…Thus, we optimized the preparation conditions to permit droplet transfer at a much lower concentration of sucrose (approx. 1.5% w/v) that hardly affects WGE translation (Takahashi and Ogawa, 2020).…”

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confidence: 99%

“…Such high amounts of sucrose severely inhibit gene expression, one of the most important biochemical reactions (Elani et al, 2015). For example, eukaryotic cell-free translation based on wheat germ extract (WGE) is suppressed by more than 80% in the presence of 10% w/v sucrose (Takahashi and Ogawa, 2020). Thus, we optimized the preparation conditions to permit droplet transfer at a much lower concentration of sucrose (approx.…”

mentioning

confidence: 99%

A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free Translation in the Interior

Takahashi¹,

Ogawa²

2021

BIO-PROTOCOL

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Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes, the spontaneous emulsion transfer method is superior to others in that it allows us to readily prepare relatively large liposomes whose sizes are controlled (from micrometer-to millimeter-sized liposomes) without special equipment. However, conventional protocols for this method require liposomes to contain a considerably high concentration of sucrose (high-density solute), which severely inhibits gene expression, one of the most important biochemical reactions. Thus, we optimized the preparation conditions to develop a wheat germ extract (WGE)-based protocol that requires a much lower concentration of sucrose and has almost no effect on eukaryotic cell-free translation. Our protocol allows us to successfully prepare millimeter-sized, moderately stable, WGE-encapsulating liposomes in which WGE translation takes place efficiently. Since a broad range of genes derived from various types of organisms can be efficiently translated in a WGE-based translation system, liposomes prepared using our protocol would be useful as a versatile research tool for artificial cells.

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Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer

Cited by 7 publications

References 22 publications

Molecular Cybernetics: Challenges toward Cellular Chemical Artificial Intelligence

Molecular Cybernetics: Challenges toward Cellular Chemical Artificial Intelligence

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane

A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free Translation in the Interior

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