preparation of extracts and t7 rna polymerase for cell-free protein synthesis systems

<meta charset="utf8"></head><body>CFPS-protokoll för syntetiska celler i mikrofack

The synthesis of synthetic or artificial cells has emerged as a highly prominent field of interdisciplinary research, attracting substantial interest from scientists across the domains of synthetic biology, chemistry, and biophysics. These scientists are united by the common goal of constructing a minimal living cell1,2,3. The rapid growth of this field has been in step with significant advancements in critical technologies, such as recombinant DNA manipulation4, biomimetic materials5, and microfabrication techniques for compartmentalization6, including the Cell-Free Protein Synthesis (CFPS) method7. CFPS systems encompass the essential cellular machinery for transcription and translation, providing the foundational framework for the development and integration of multifunctional artificial cells.
Although CFPS techniques are frequently used in the assembly of synthetic cells, developing a robust and tailored CFPS system for the assembly of various synthetic cell systems remains a complex challenge. Currently, numerous CFPS systems are available, derived from both prokaryotes and eukaryotes model organisms8, each specialized for particular applications in synthetic cell synthesis. Beyond their central roles in transcription and translation, CFPS systems vary in their main components and associated preparation procedures. These variations, which include differences in cell extracts, RNA polymerases, template preparation methods, and buffer compositions, are largely due to the distinct development trajectories pursued by research groups that have intensively optimized their systems for maximal protein yield.
Among the various components of the CFPS system, the cell extract is a critical enzymatic pool for transcription and translation, and thus a key determinant of CFPS performance9. Escherichia coli (E. coli)-based CFPS is the most commonly utilized system due to its status as the best-understood prokaryotic organism. Furthermore, a fully reconstituted CFPS system comprising individually purified proteins and ribosomes, known as PURE10, has been developed by Ueda&#39;s research group, which is particularly suited for applications requiring a clear background. Today, even E. coli-based CFPS systems have diversified, especially in terms of the source strains for the extrac11 and methods of preparation12,13, RNA polymerase14,15, energy sources16,17, and buffer systems18,19. The most frequently used strains include K12 and B strain derivatives, such as A1920, JM10921, BL21 (DE3)22, and Rossetta223, alongside their genetically modified counterparts.
Initially, E. coli strains with reduced RNase and protease activities were chosen to enhance mRNA stability and the stability of newly synthesized recombinant proteins, leading to increased final protein yields24. Subsequently, E. coli extracts were engineered to facilitate specific post-translational modifications, including glycosylation25, phosphorylation26, and lipidation27, were developed to achieve the above posttranslational modifications. Additionally, an array of additives such as molecular chaperons28 and chemical stabilizers have been incorporated to aid the folding of target proteins, contributing to the diversification of CFPS systems. The bacteriophage T7 RNA polymerase, known for its high processivity, is predominantly employed for transcription, although other polymerases such as SP629 have also been utilized. E. coli endogenous RNA polymerase has been adapted for the prototyping of genetic circuits leveraging sigma factors30. Lastly, a variety of energy precursors31,32,33 and different salts and buffer components19,34,35 have been systematically optimized to enhance productivity.
Besides the CFPS system itself, the encapsulation methods as well as compartmentalization materials are also vital for the successful synthetic cell assembly. Various systems that have been developed to successfully encapsulate the CFPS reaction include surfactant-stabilized water/oil droplets, lipid/polymer, and their hybrid unilamellar vesicles (with diameters ranging from 50 nm to several &#956;m), as well as planar-supported lipid bilayers. However, due to the complexed molecule content of the CFPS system, the success rate of encapsulation depends on specific cases, particularly for the formation of vesicles. To improve the success rate and efficiency of encapsulation of CFPS, various microfluid chips have been developed to facilitate the formation of both droplets and vesicles36. Nevertheless, additional chips and devices will need to be established.
This protocol delineates an E. coli CFPS system utilizing the BL21(DE3) strain, which is a commonly employed host for recombinant protein production. The protocol encompasses a detailed account of the cell extract preparation, template preparation, and standard expression optimization using a fluorescent reporter protein. Moreover, we present exemplary outcomes achieved by encapsulating the CFPS system within diverse micro-compartments, including monolayer droplets, double emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we expound upon the pivotal procedural elements and the requisite conditions indispensable for the successful establishment of these CFPS systems within distinct environmental contexts.

<meta charset="utf8"></head><body>Författare i fokus: Optimering av CFPS-system för konstruktion av syntetiska celler

Cellfritt proteinsyntessystem för att bygga syntetiska celler

All our research focuses on cell-free synthetic biology. This involve bottom up assembly synthetic cells, functional and structured analysis of membrane proteins, protein engineering while incorporation of non conical amino acids and cell-free metabolic engineering. Currently besides cell-free protein synthesis, micro for edicts device by micro fabrication through soft lithography development of biocompatible materials and the general AI at the forefront technologies that drives the faster development of cell-free synthetic biology.The configuration presented as a protocol establishes a straightforward, yet robust system that is well shooted various micro compartment environments, including the Jove-ase, GOase, SL-based, that we should in our representative results. In the future, we will focus on the development of tailored cell-free systems, which enable the assembly and integration of different modular cell mimicry systems towards the ultimate goal of building a minimal cell.

Framställning av extrakt och T7 RNA-polymeras för cellfria proteinsyntessystem

preparation-extracts-t7-rna-polymerase-for-cell-free-protein

Research

JoVE Journal

Biochemistry

Preparation of Extracts and T7 RNA Polymerase for Cell-Free Protein Synthesis Systems

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

To begin, obtain pre-cultured cells and wash them two times with 35 milliliters of pre-chilled S30 buffer A.Pellet the cells. And re-suspend them in S30 buffer B.For disrupting the cells, transfer the cell suspension to a French press metal disruption chamber and secure it in the hydraulic platform with the safety lock. Turn on the hydraulic pump and start the disruption.Control the outlet valve to collect the cell suspension in a new 50-milliliter tube, maintaining the pressure at 17, 000 pounds per square inch. Centrifuge the resulting lysate at 30, 000G for 30 minutes at four degrees Celsius and collect the supernatant. Centrifuge again and incubate the collected supernatant with 0.3 volume of pre-incubation buffer with gentle shaking at 37 degrees Celsius for 80 minutes.Dialyze the resulting runoff mixture for two hours against two liters of S30 buffer C overnight at four degrees Celsius. Centrifuge the dialyzed sample and aliquot the supernatant. Flash freeze the samples immediately in liquid nitrogen.After pelleting the cultured cells, re-suspend them in 30 milliliters of T7 buffer A.Disrupt the cells in one pass through the French press at 15, 000 pounds per square inch, and centrifuge the lysate to remove the cell debris. Add streptomycin sulfate drop by drop to the supernatant to reach a final concentration of 4%After a short incubation on ice, centrifuge the lysate as before. Load the filtered samples onto a strong anion exchange column.After washing the column, elute the sample with 10 column volumes of T7 buffer C at a flow rate of three milliliters per minute. Add glycerol to reach a final concentration of 10%and subject the resulting mixture to ultrafiltration to obtain a final concentration of three to four milligrams per milliliter.

Lipid Encapsulation Procedure to Obtain Cell-Free Protein Synthesis System

To begin, obtain a surfactant containing fluorinated, or a lipid mineral oil solution. Add cell-free protein synthesis or CFPS mixture to 500 microliters of the previously prepared oil in a 1.5 milliliter tube, and rub the tube vigorously on the tube rack 50 times to form fine droplets. For giant unilamellar vesicle, or GUV preparation, add 57 microliters of chloroform into a four milliliter glass vial, followed by 18 microliters of lipids, to a final concentration of eight millimolar.Evaporate the chloroform under an argon flow for 15 minutes, and then under vacuum for one hour. Dissolve the resulting dry lipids in 1, 500 microliters of mineral oil to reach a final concentration of 400 micromolar. To obtain an interfacial lipid layer, slowly layer two 50 microliters of lipid mineral oil solution on top of 500 microliters of the outer solution, taken in a 1.5 milliliter tube.Incubate at room temperature for 30 minutes to form a stable interfacial lipid layer. For the inner solution, add T7 RNA polymerase S30 extract, and DNA template into the cold pre inner solution. Mix 50 microliters of inner solution, and 500 microliters of lipid mineral oil, and vortex vigorously.Leave the tube on ice for 10 minutes, and slowly add 20 microliters of this emulsion mixture to the top of the previously prepared oil phase in the 1.5 milliliter tube. Centrifuge at about 800 G for 10 minutes at four degrees celsius. And observe the formation of GUVs at the bottom of the tube.After removing the upper oil phase, aspirate 30 microliters of GUVs from the bottom of the tube carefully. Add seven drops of sulfuric acid, and two drops of 50%hydrogen peroxide to the center of a cover slide, and incubate for 45 minutes. After rinsing thoroughly with ultrapure water, glue it to a cut 0.5 milliliters microfuge tube.Cure under an ultraviolet lamp for 10 minutes to form a reaction chamber. Next, add 75 microliters of the small unilamellar vesicles, or SUV suspension to the preformed reaction chambers, and incubate it on a heat block at 37 degrees celsius for one minute. Then, add 150 microliters of supported lipid bilayer, or SLB buffer A to the reaction chamber for an additional two minute incubation.Wash the chamber with hundred microliters of SLB buffer B prior to a buffer exchange to the S 30 buffer C, leaving 100 microliters of buffer inside the chamber to prevent the form's supported lipid bilayer from drying out. Using confocal laser scanning microscopy, the expression of green fluorescent protein and mCherry encapsulated in droplets was successfully captured. The fluorescence image of encapsulated CFPS within GUVs showed expression of super folder green fluorescent protein within the labeled lipid bilayer.