<meta charset="utf8"></head><body>用于微区室化合成细胞的 CFPS 方案

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine(DOPC)</td><td>Avanti</td><td>850375P</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-L-serine (sodium salt)(DOPS)</td><td>Avanti</td><td>840035P</td><td></td></tr><tr><td>1,4 dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>1.11474</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td><td>Avanti</td><td>850457P</td><td></td></tr><tr><td>3,5-cyclic AMP (cAMP)</td><td>Sigma-Aldrich</td><td>A9501</td><td></td></tr><tr><td>50 mL tubes</td><td>Eppendorf</td><td>Eppendorf Tubes BioBased</td><td></td></tr><tr><td>50% hydrogen peroxide</td><td>Sigma-Aldrich</td><td>516813</td><td></td></tr><tr><td>Acetate</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Agar powder</td><td>Sigma-Aldrich</td><td>05040</td><td></td></tr><tr><td>Alanin</td><td>Sigma-Aldrich</td><td>A4349</td><td></td></tr><tr><td>Amicon Stirred Cells</td><td>MerckMillipore</td><td>UFSC05001</td><td></td></tr><tr><td>Ammonium acetate (NH4OAc)</td><td>Sigma-Aldrich</td><td>A7262</td><td></td></tr><tr><td>Arginin</td><td>Sigma-Aldrich</td><td>A4474</td><td></td></tr><tr><td>Asparagin</td><td>Sigma-Aldrich</td><td>A0884</td><td></td></tr><tr><td>Aspartat</td><td>Sigma-Aldrich</td><td>A5474</td><td></td></tr><tr><td>ATP</td><td>Roche</td><td>11140965001</td><td></td></tr><tr><td>Atto 488 DOPE</td><td>Sigma-Aldrich</td><td>67335</td><td></td></tr><tr><td>Atto 647N DOPE</td><td>Sigma-Aldrich</td><td>42247</td><td></td></tr><tr><td>Baffled Erlenmeyer flask</td><td>Shuniu</td><td>250 mL, 1000mL</td><td></td></tr><tr><td>Bovine Serum Albumin(BSA)</td><td>Roche</td><td>10711454001</td><td></td></tr><tr><td>Centrifugetube</td><td>Eppendorf</td><td>Eppendorf Tubes 3810X</td><td></td></tr><tr><td>Centrifugetube rack</td><td>Eppendorf</td><td>0030119819</td><td></td></tr><tr><td>Chemiluminescence and epifluorescence imaging system</td><td>Uvitec</td><td>Alliance Q9 Advanced</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306</td><td></td></tr><tr><td>Confocal Laser Scanning Microscopy (LSM)</td><td>ZEISS</td><td>LSM 780</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Thermo Fisher Scientific</td><td>C10283</td><td></td></tr><tr><td>Coverslip</td><td>Thermo Scientific</td><td>Menzel BB02400500A113MNZ0</td><td></td></tr><tr><td>creatine kinase (CK)</td><td>Roche</td><td>10127566001</td><td></td></tr><tr><td>Creatine phosphate (CP)</td><td>Sigma-Aldrich</td><td>10621714001</td><td></td></tr><tr><td>Culture dish</td><td>Huanqiu</td><td>90 mm</td><td></td></tr><tr><td>Cystein</td><td>Sigma-Aldrich</td><td>C5360</td><td></td></tr><tr><td>Cytidine 5&#039;-triphosphate disodium salt (CTP)</td><td>aladdin</td><td>C101487</td><td></td></tr><tr><td>Dialysis membrane</td><td>Spectrum</td><td>Standard RC Tubing MWCO: 12-14 kD</td><td></td></tr><tr><td>E.Z.N.A. Cycle Pure Kit</td><td>Omega Bio-Tek</td><td>D6492-01</td><td></td></tr><tr><td>Electro-Heating Standing-Temperature Cultivator</td><td>Yiheng instrument</td><td>DHP-9602</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid(EDTA)</td><td>Biosharp</td><td>1100027</td><td></td></tr><tr><td>Fluorescent plate reader</td><td>BioTek</td><td>Synergy 2</td><td></td></tr><tr><td>Fluorinated oil</td><td>Suzhou CChip scientific instrument</td><td>2%HFE7500</td><td></td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich</td><td>47612</td><td></td></tr><tr><td>French Press</td><td>G.Heinemann</td><td>HTU-DIGI-Press</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glutamat</td><td>Sigma-Aldrich</td><td>G5667</td><td></td></tr><tr><td>Glutamin</td><td>Sigma-Aldrich</td><td>G5792</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Glycin</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate(GTP)</td><td>Roche</td><td>10106399001</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HiPrep Q FF 16/10</td><td>Cytiva</td><td>28936543</td><td></td></tr><tr><td>Histidin</td><td>Sigma-Aldrich</td><td>H6034</td><td></td></tr><tr><td>Isoleucin</td><td>Sigma-Aldrich</td><td>I5281</td><td></td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P8281</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Leucin</td><td>Sigma-Aldrich</td><td>L6914</td><td></td></tr><tr><td>Lysin</td><td>Sigma-Aldrich</td><td>L5501</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate (Mg(OAc)&lt;sub&gt;2&lt;/sub&gt; )</td><td>Sigma-Aldrich</td><td>M5661</td><td></td></tr><tr><td>Magnesium chloride(MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Methionin</td><td>Sigma-Aldrich</td><td>M8439</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424 R</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M5904</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell Systems</td><td>Bio-Rad</td><td>1645050</td><td></td></tr><tr><td>Multipurpose Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>NaN3</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Nucleic Acid &amp;amp; Protein UV-Assay Measurements</td><td>IMPLEN</td><td>NanoPhotometer N60</td><td></td></tr><tr><td>NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA</td><td>MACHEREY-NAGEL</td><td>740414.5</td><td></td></tr><tr><td>Nunc-Immuno MicroWell 96 well polystyrene plates</td><td>Sigma-Aldrich</td><td>P8616</td><td></td></tr><tr><td>PCR Thermal Cycler</td><td>Eppendorf</td><td>Mastercycler nexus</td><td></td></tr><tr><td>Peptone</td><td>Sigma-Aldrich</td><td>83059</td><td></td></tr><tr><td>Phenylalanin</td><td>Sigma-Aldrich</td><td>P8740</td><td></td></tr><tr><td>Phosphoenolpyruvat (PEP)</td><td>GLPBIO</td><td>GC44635</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>PMSF-RO</td><td></td></tr><tr><td>Polyethylene glycol 8000 (PEG 8000)</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>Potassium Acetate(KOAc)</td><td>Sigma-Aldrich</td><td>P5708</td><td></td></tr><tr><td>Potassium chloride(KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Potassium glutamate (K-glutamate)</td><td>Sigma-Aldrich</td><td>G1501</td><td></td></tr><tr><td>Potassium hydroxide(KOH)</td><td>Sigma-Aldrich</td><td>221473</td><td></td></tr><tr><td>Prolin</td><td>Sigma-Aldrich</td><td>P8865</td><td></td></tr><tr><td>Pyruvate kinase (PK)</td><td>Sigma-Aldrich</td><td>P9136</td><td></td></tr><tr><td>Serin</td><td>Sigma-Aldrich</td><td>S4311</td><td></td></tr><tr><td>Shaker</td><td>Zhichushakers</td><td>ZQZY-AF8</td><td></td></tr><tr><td>Sodium chloride(NaCl)</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sodium hydroxide(NaOH)</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sucrose</td><td>aladdin</td><td>S112226</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td></td></tr><tr><td>Syringe Filters</td><td>Jinteng</td><td>0.45 &amp;mu;m</td><td></td></tr><tr><td>Test tube</td><td>Shuniu</td><td>20 mL</td><td></td></tr><tr><td>TGX FastCast Acrylamide Kit, 12%</td><td>Bio-Rad</td><td>#1610175</td><td></td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>ThermoMixer C</td><td></td></tr><tr><td>Threonin</td><td>Sigma-Aldrich</td><td>T8441</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>V900483</td><td></td></tr><tr><td>tRNA</td><td>Roche</td><td>10109550001</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293</td><td></td></tr><tr><td>Tryptophan</td><td>Sigma-Aldrich</td><td>T8941</td><td></td></tr><tr><td>Tyrosin</td><td>Sigma-Aldrich</td><td>T8566</td><td></td></tr><tr><td>UTP Trisodium salt (UTP)</td><td>aladdin</td><td>U100365</td><td></td></tr><tr><td>Vacuum Pump with Circulated Water System</td><td>Zhengzhou Greatwall Scientific Industrial and Trade Co.Ltd</td><td>SHB-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66626/66626eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Valin</td><td>Sigma-Aldrich</td><td>V4638</td><td></td></tr><tr><td>Vortex Mixers</td><td>Kylin-Bell</td><td>Vortex QL-861</td><td></td></tr><tr><td>Water purification system</td><td>MerckMillipore</td><td>Direct ultrapure water (Type 1)</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>70161</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>444203</td><td></td></tr></tbody></table>

cell-free protein synthesis system for building synthetic cells

Read this Scientific Article Protocol about Cell-free Protein Synthesis System for Building Synthetic Cells at JoVE.com

<meta charset="utf8"></head><body>作者聚焦：优化 CFPS 系统以进行合成细胞构建

用于构建合成细胞的无细胞蛋白质合成系统

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 ...

cell-free-protein-synthesis-system-for-building-synthetic-cells

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Cell-Free Protein Synthesis System for Building Synthetic Cells

1.7K 次观看.  Jiangsu Normal University. 我们所有的研究都集中在无细胞合成生物学上。这涉及自下而上的组装合成细胞、膜蛋白的功能和结构化分析、蛋白质工程，同时掺入非锥形氨基酸和无细胞代谢工程。目前，除了无细胞蛋白质合成外，micro for edict 通过软光刻技术通过微制造来制造生物相容性材料的开发，以及推动无细胞合成生物学更快发展的前沿技术的通用 AI。作为协议呈?...

视频: 作者聚焦：优化 CFPS 系统以进行合成细胞构建

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

科研

生物工程

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

化学

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

一锅纯无细胞系统

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

使用无细胞自动诱导工作流程在24小时内从细胞到无细胞蛋白质合成

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.

利用线性DNA模板从核酸外切酶缺陷细胞提取物中合成无细胞蛋白

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...