upscaling a metabolic network for micrometer&#45;sized vesicles

Protocole d’encapsulation de vésicules lipidiques pour des réseaux protéiques complexes

The field of bottom-up synthetic biology focuses on constructing (minimal) cells1,2 and metabolic bioreactors for biotechnological3,4 or biomedical purposes5,6,7,8. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical functions of proteins and reaction networks9. As an intermediate step towards an autonomously functioning synthetic cell, modules are developed that capture essential features of living cells such as metabolic energy conservation, protein and lipid synthesis, and homeostasis. Such modules not only enhance our understanding of life but also have potential applications in the fields of medicine8 and biotechnology10.
Transmembrane proteins are at the heart of virtually any metabolic network as they transport molecules in or out of the cell, signal, and respond to the quality of the environment, and play numerous biosynthetic roles. Thus, the engineering of metabolic modules in synthetic cells requires in most cases the reconstitution of integral and/or peripheral membrane proteins into a membrane bilayer composed of specific lipids and high integrity (low permeability). The handling of these membrane proteins is challenging and requires specific knowledge and experimental skills.
Several methods have been developed to reconstitute membrane proteins within phospholipid vesicles, most often with the purpose of studying the function11,12, regulation13, kinetic properties14,15, lipid dependence15,16, and/or stability17 of a specific protein. These methods involve the rapid dilution of detergent-solubilized protein into aqueous media in the presence of lipids18, the removal of detergents by incubating detergent-solubilized protein with detergent-destabilized lipid vesicles and absorption of the detergent(s) onto polystyrene beads19, or the removal of detergents by dialysis or size-exclusion chromatography20. Organic solvents have been used to form lipid vesicles, for example, via the formation of oil-water interphases21, but the majority of integral membrane proteins are inactivated when exposed to such solvents.
In our laboratory, we mostly reconstitute membrane proteins by the detergent-absorption method to form large-unilamellar vesicles (LUVs)19. This method allows the co-reconstitution of multiple membrane proteins and the encapsulation in the vesicle lumen of enzymes, metabolites, and probes22,23. The membrane protein-containing LUVs can be converted into giant-unilamellar vesicles (GUVs) with/without encapsulation of water-soluble components, using either electroformation24 or gel-assisted swelling25 and specific conditions to preserve the integrity of the membrane proteins26.
This paper presents a protocol for the reconstitution in LUVs of an out-of-equilibrium metabolic network that regenerates ATP through the breakdown of L-arginine into L-ornithine27. The formation of ATP is coupled to the production of glycerol-3-phosphate (G3P), an important building block for phospholipid synthesis22,28. The metabolic pathway consists of two integral membrane proteins, an arginine/ornithine (ArcD) and a G3P/Pi antiporter (GlpT). In addition, three soluble enzymes (ArcA, ArcB, ArcC) are required for the recycling of ATP, and GlpK is used to convert glycerol into glycerol 3-phosphate, using the ATP from the breakdown of L-arginine, see Figure 1 for a schematic overview of the pathway. This protocol represents a good starting point for the future construction of even more complex reaction networks-for the synthesis of lipids or proteins or the division of cells. The lipid composition of the vesicles supports the activity of a wide variety of integral membrane proteins and has been optimized for the transport of diverse molecules into or out of the vesicles27,29,30.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66627/66627fig01v2.jpg" /> 
Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In short, purified membrane proteins (solubilized in dodecyl-&#946;-D-maltoside, DDM) are added to preformed lipid vesicles that have been destabilized with Triton X-100, which allows the insertion of the proteins into the membrane. The detergent molecules are subsequently (slowly) removed by the addition of activated polystyrene beads, resulting in the formation of well-sealed proteoliposomes. Soluble components can then be added to the vesicles and encapsulated via freeze-thaw cycles, which traps the molecules in the process of membrane fusion. The obtained vesicles are highly heterogeneous and many are multilamellar. They are then extruded through a polycarbonate filter with a pore size of 400, 200, or 100 nm, which yields more uniformly sized vesicles; the smaller the pore size, the more homogeneous and unilamellar the vesicles but at the price of a smaller internal volume. Non-incorporated proteins and small molecules are removed from the external solution by size-exclusion chromatography. The proteoLUVs can be converted into micrometer size vesicles by gel-assisted swelling, and these proteoGUVs are then collected and trapped in a microfluidic chip for microscopic characterization and manipulation. Figure 2 shows a schematic overview of the full protocol.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66627/66627fig02v2.jpg" /> 
Figure 2: Overview of the protocol for reconstituting membrane proteins and encapsulating enzymes and water-soluble components in lipid vesicles of sub-micrometer (LUVs) and micrometer size (GUVs). <a href="https://www.jove.com/files/ftp_upload/66627/66627fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The reconstitution and encapsulation protocols work well and the functionality of the proteins is retained, but the proteoLUVs and proteoGUVs are heterogeneous in size. Microfluidic approaches31,32 allow the formation of micrometer-sized vesicles that are more homogeneous in size, but functional reconstitution of membrane proteins is generally not possible because residual solvent in the bilayer inactivates the proteins. The proteoLUVs range in size from 100 to 400 nm, and at low concentrations of enzymes, the encapsulation may lead to vesicles with incomplete metabolic pathways (stochastic effects; see Figure 3). LUVs are ideal for constructing specific metabolic modules, as shown here for the production of ATP and building blocks like G3P. Such proteoLUVs can potentially be encapsulated in GUVs and serve as organelle-like compartments for the host vesicles.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66627/66627fig03v2.jpg" /> 
Figure 3: Number of molecules per vesicle with a diameter of 100, 200, or 400 nm. (A) When the encapsulated proteins (enzymes, probes) are in the range of 1-10 &#181;M. (B) The reconstitution is done at 1 to 1,000, 1 to 10,000, and 1 to 100,000 membrane proteins per lipid (mol/mol). We make the assumption that molecules are encapsulated at the indicated concentrations and incorporated in the membrane at these protein-to-lipid ratios. For some enzymes, we have seen that they bind to membranes, which can increase their apparent concentration in the vesicles. Abbreviation: LPR = Lipid-Protein-Ratio <a href="https://www.jove.com/files/ftp_upload/66627/66627fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Pleins feux sur l’auteur : Relever les défis de l’ingénierie des cellules synthétiques

Construction de réseaux métaboliques hors équilibre dans des vésicules de taille nanométrique et micrométrique

In the Netherlands, the BaSyC consortium aims to construct synthetic cells from molecular components. The BaSyC consortium is composed of research groups ranging from molecular biology to chemistry, physics, and computational sciences. The goal of this consortium is to create a self-replicating system capable of autonomous growth to transmit information and to divide.And we currently focus on the building of molecular networks that are integrated towards more complex molecular systems. Recent developments in synthetic biology include the engineering of modules for cellular functions, integrating them into complex networks for applications in diagnostics, therapy, synthetic immunology, and adaptive materials. This progress spans groups in Europe, America, and Asia, advancing both top-down and bottom-up approaches to synthetic cell construction.Current technologies in our field include the engineering and evolutionary methods to construct and optimize metabolic networks. We use AI for molecular selection and advanced biochemical and biophysical techniques for component isolation and network formation. We also employ membrane reconstitution, physical encapsulation, and visualization via fluorescent spectroscopy and microscopy.With the integration of modules, the number of membrane proteins and enzymes increase enormously, which poses constraints on our reconstitution and encapsulation technologies. Stochastic encapsulation becomes an issue with sub-micron size vesicles, and the efficiency of forming micrometer-size vesicles decreases when multiple membrane proteins are reconstituted in the lipid membrane. In Groningen, we have developed metabolic networks for ATP productions and maintaining an out-of-equilibrium state through substrate feeding and product export.Additionally, we linked ATP recycling to the synthesis and excretion of lipid precursors, enabling secondary vesicles to produce lipids, which is a significant advancement in our field.

Mise à l’échelle d’un réseau métabolique pour des vésicules de taille micrométrique

Pour commencer, insérez le bas d’une pointe de pipette coupée de 200 microlitres dans un dispositif de piégeage microfluidique préfabriqué. À l’aide d’une seringue équipée d’un filtre de 0,2 micromètre, injectez 400 microlitres de solution de caséine bêta dans le réservoir d’entrée de la puce. Centrifugez la puce à 900 G pendant six minutes.Le niveau de liquide doit maintenant être égal à l’entrée et à la sortie. Dessinez le contour de l’entretoise sur deux diapositives d’objectif. Nettoyez les lames avec un plasma à haute teneur en oxygène pendant une minute pour les rendre hydrophiles.Ajoutez de l’agarose à basse température de gélification sur le dessus de la lame jusqu’à ce qu’elle soit entièrement recouverte d’agarose. Ensuite, inclinez la glissière à un angle de 90 degrés et égouttez l’excès d’agarose sur un mouchoir. Placez les lames à 50 degrés Celsius pendant 30 minutes.À l’aide d’un sonificateur à sonde portatif, équipé d’une sonde d’un millimètre, sonicez les protéoliposomes préparés. Gardez les SUV proteo sur la glace pendant 30 secondes avant de répéter la sonication cinq fois. À l’aide d’une seringue de 100 microlitres, déposez des gouttelettes de 0,5 microlitre des SUV proteo sur un gel d’agarose préparé.Utilisez un flux d’azote pendant environ 10 minutes pour sécher les gouttelettes. À l’aide d’une seringue et d’une aiguille, pipetez la solution gonflante dans une chambre préparée à travers un petit trou sur le côté et laissez les vésicules gonfler à 22 degrés Celsius pendant au moins 30 minutes. Tapotez la chambre sur une surface solide pour récolter les GUV du gel.Pour piéger les GUV pour la microscopie, montez une puce microfluidique passivée sur un microscope. Après avoir vérifié s’il y a des défauts, connectez le tube avec une aiguille pliée à la sortie du réservoir. Après avoir connecté l’autre extrémité à une seringue d’un millilitre, montez la seringue sur une pompe avec un débit maximal de 10 microlitres par minute.Remplacez le tampon de lavage du réservoir par un fluide frais. Laver avec au moins 80 microlitres de tampon P.Après avoir retiré l’excès de tampon, ajoutez les proteo GUVs dans le réservoir. Surveillez la puce au fil du temps jusqu’à ce qu’un nombre suffisant de GUV aient été piégés dans la puce.Pipetez l’excès de solution GUV. Ajoutez ensuite le tampon de lavage P à un débit de 0,1 à un microlitre par minute. Enfin, localisez les pièges avec une quantité suffisante de vésicules.Après avoir appliqué les paramètres au microscope, ajoutez le tampon R dans le réservoir à un débit de 0,5 microlitre par minute. La réhydratation de LUV protéo séchés sur un gel d’agarose à basse température de gélification a entraîné la formation de vésicules de taille micrométrique. Les GUV ont été récoltés et piégés dans un dispositif microfluidique passivé à la caséine bêta.

upscaling-a-metabolic-network-for-micrometersized-vesicles

Research

JoVE Journal

Biochemistry

Upscaling a Metabolic Network for Micrometer&#45;Sized Vesicles

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Recherche

Biochimie

Reconstitution of Purified Membrane Proteins into Preformed Liposomes

Reconstitution de protéines membranaires purifiées en liposomes préformés

Encapsulation of a Metabolic Network for ATP Recycling and Glycerol 3&#45;P Synthesis in Submicron&#45;Size Vesicles

Encapsulation d’un réseau métabolique pour le recyclage de l’ATP et la synthèse du glycérol 3-P dans des vésicules de taille submicronique