この材料は (v. n.) に海軍研究所受賞番号 N00014-13-1-0074 によってサポートされる作業に基づいて、人間科学フロンティア許可番号 RGP0037/2015 (砲兵/ベトナム) と二国間の科学技術振興財団 (v. n.) に 2014400 を与えます。

<ol>
	<li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol Adv. , (2011).</li><li>Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+synthetic+biology:+Thinking+outside+the+cell.">Cell-free synthetic biology: Thinking outside the cell.</a> Metab Eng. , (2011).</li><li>Caschera, F., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+2.3+mg/mL+of+protein+with+an+all+Escherichia+coli+cell-free+transcription-translation+system.">Synthesis of 2.3 mg/mL of protein with an all Escherichia coli cell-free transcription-translation system.</a> Biochimie. 99, 162-168 (2014).</li><li>Shin, J., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+E.+coli+cell-free+expression+toolbox:+application+to+synthetic+gene+circuits+and+artificial+cells.">An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells.</a> ACS Synth Biol. 1 (1), 29-41 (2012).</li><li>Chemla, Y., Ozer, E., Schlesinger, O., Noireaux, V., Alfonta, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+expanded+cell-free+protein+synthesis+using+endogenous+pyrrolysyl+orthogonal+translation+system.">Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system.</a> Biotechnol Bioeng. , (2015).</li><li>Hong, S. H., Kwon, Y. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-standard+amino+acid+incorporation+into+proteins+using+Escherichia+coli+cell-free+protein+synthesis.">Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis.</a> Frontiers in Chemistry. 2, (2014).</li><li>Noireaux, V., Bar-Ziv, R., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cell-free+genetic+circuit+assembly.">Principles of cell-free genetic circuit assembly.</a> Proc Natl Acad Sci U S A. 100 (22), 12672-12677 (2003).</li><li>Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+DNA+for+Rapid+Prototyping+of+Synthetic+Biological+Circuits+in+an+Escherichia+coli+Based+TX-TL+Cell-Free+System.">Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System.</a> Acs Synthetic Biology. 3 (6), 387-397 (2014).</li><li>Takahashi, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+prototyping+genetic+networks+with+cell-free+transcription-translation+reactions.">Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.</a> Methods. , (2015).</li><li>Shin, J., Jardine, P., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Replication,+Synthesis,+and+Assembly+of+the+Bacteriophage+T7+in+a+Single+Cell-Free+Reaction.">Genome Replication, Synthesis, and Assembly of the Bacteriophage T7 in a Single Cell-Free Reaction.</a> ACS Synthetic Biology. 1 (9), 408-413 (2012).</li><li>Garamella, J., Marshall, R., Rustad, M., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+All+E.+coli+TX-TL+Toolbox+2.0:+A+Platform+for+Cell-Free+Synthetic+Biology.">The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.</a> ACS Synth Biol. , (2016).</li><li>Karzbrun, E., Shin, J., Bar-Ziv, R. H., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+dynamics+of+protein+synthesis+in+a+cell-free+system.">Coarse-grained dynamics of protein synthesis in a cell-free system.</a> Phys Rev Lett. 106 (4), 048104 (2011).</li><li>Daube, S. S., Arad, T., Bar-Ziv, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+co-synthesis+of+protein+nanoassemblies:+tubes,+rings,+and+doughnuts.">Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts.</a> Nano Lett. 7 (3), 638-641 (2007).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunoblot+assay+reveals+that+bacteriophage+T4+thymidylate+synthase+and+dihydrofolate+reductase+are+not+virion+proteins.">An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.</a> J Virol. 69 (4), 2119-2125 (1995).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> J Vis Exp. (79), e50762 (2013).</li><li>Shin, J. N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cell-free+expression+with+the+endogenous+E.+coli+RNA+polymerase+and+sigma+factor+70.">Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70.</a> J Biol Eng. , (2010).</li><li>Caschera, F., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+amino+acid+mixtures+for+cell-free+expression+systems.">Preparation of amino acid mixtures for cell-free expression systems.</a> Biotechniques. 58 (1), 40-43 (2015).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+biochemical+reactions+within+cells+differ+from+those+in+test+tubes.">How can biochemical reactions within cells differ from those in test tubes.</a> J Cell Sci. 119, 2863-2869 (2006).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+macromolecular+crowding+for+protein+assembly.">Implications of macromolecular crowding for protein assembly.</a> Curr Opin Struct Biol. 10 (1), 34-39 (2000).</li><li>Zimmerman, S. B., Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding:+biochemical,+biophysical,+and+physiological+consequences.">Macromolecular crowding: biochemical, biophysical, and physiological consequences.</a> Annu Rev Biophys Biomol Struct. 22, 27-65 (1993).</li></ol>

著者宣言次競合金融利益のため: Noireaux 研究所は、MYcroarray、MYtxtl 無細胞タンパク質発現キットのディストリビューターから研究資金を受け取る。

次の陳らの手法14 SPMC、重複感染の適切な条件を決定する際の重要なステップは達されます。最も密接にホスト株の重複感染に耐える能力を制御するパラメーターは頻繁に感染しているバクテリオファージの初期濃度です。宿主細胞は、バクテリオファージの非常に少量の初期感染前に対数増殖期でなければなりません。最終的には、バクテリオファージはまた対数増...

過去 10 年間無料のセルの表現技術を創発的学際的な研究領域合成と定量的に関連する生物学の新しいアプリケーションに対応し設計されています。もともと独立した生きている有機体の蛋白質を表現するために使用、新しい携帯無料 TXTL システムは両方基本的な応用科学1、2、かなりこの技術の範囲を広げるために開発されています。TXTL プラットフォームの新世代はユーザーフレンドリーをできるように設計されている効率的 (到達するとバッチ モード3におけるタンパク質合成の 2 mg/mL) より転写4のレベルで汎用性とするようモジュールを簡単に統合自然小説または既存の生物学的システム5,6の機能を拡張する合成関数。特に、携帯無料 TXTL システムとなっている規制要素または小さな遺伝的回路7,8,9などの遺伝的プログラムのラピッドプロトタイピングの便利なデザイン、ビルド、テストを減らすことによって数日サイクルします。驚くことに、新しい TXTL システムは、coliphages10,11, アクティブなゲノムの再構成をサポートする十分な性能を示す強いの完全な合成など大規模な DNA プログラムを処理できます。DNA は、生き物のエンティティがエンコードされます。
TXTL システムは、従来の in vitro建設的な生化学的アッセイに比べて多くの技術的な優位性を提示します。携帯無料 TXTL は、遺伝子発現のプロセスを生きている細胞の複雑な細胞質とは対照的削減とオープン環境で最終製品にリンクします。TXTL では、DNA を利用して、現代 DNA のアセンブリ技術と手頃な価格、気難しいタンパク質精製工程を必要としないに加えて高速生化学システムの in vitro、再構成します。無細胞発現分子間相互作用12の深い郭清をできるように、生化学的な反作用の部品のほとんどへの直接アクセスを提供します。TXTL 反応では、生きている細胞ではほとんど不可能で生化学と生物物理設定変更できます。これらの利点と最近の改善を考えると、TXTL 技術、合成および量的な生物学の代替プラットフォームとして人気が高まってください。それは TXTL 反応の実行に関連する適切なプラクティスを開発するためにこのようなプラットフォームを使用する方法を理解する重要な TXTL を使用して、研究コミュニティは急速に成長しているし、TXTL は、バイオ エンジニア リングの標準的な技術になって、結果の解釈。
この記事での合成、ワンポット反応でバクテリオファージのゲノムから11、MS2 などすべてのエシェリヒア属大腸菌の TXTL システムを使用する方法について説明 (RNA、3.4 kb)、ΦΧ174 (ssDNA、5.4 kb)、および T7 (dsDNA、40 kb)。どのようにファージ量合成反応 (マグネシウムとカリウム濃度) の生化学的な設定の一部に関して変更を示します。ペグ範囲 8000 濃度をエミュレート分子クラウディング バクテリオファージ合成に劇的な効果は、桁違いです。同時に転写、翻訳のプロセスを要約、関連生物学および生物物理学の基本的な質問に対処するための興味深い自己集合、単一試験管反応でこのような大規模な生化学的なシステムの実現10 (遺伝子発現制御、自己集合), だけでなく、新しいナノ構造13を構築するバクテリオファージ関数情報の転用などのアプリケーションを開発します。TXTL の実用的なに加えて我々 はプラクの試金によってバクテリオファージ増幅、ゲノム抽出と精製・ ファージ定量化メソッドを提供します。本稿では提案手法はエシェリヒア属大腸菌の抽出による無細胞システムを使用し、バクテリオファージに興味がある者に適しています。
この作品で提示プロトコルはフォローとしてまとめることができます: 1) バクテリオファージ増幅 (日 1: 接種の準備細胞、2 日目: 単一のプラーク、複数のバクテリオファージの成長と濃度、および 3 日目: バクテリオファージの浄化)、2) 二重座礁させたゲノム DNA の抽出(フェノール/クロロホルム抽出)、3) 携帯無料ファージ反応と抗体実験 (日 1: 宿主細胞をプレートし、寒天プレートを作る 2 日目: 無細胞反応とホスト細胞の前培養と一日 3: ホスト細胞文化およびバクテリオファージ価)。

<table><tbody><tr><td>Ultracentrifugation tubes</td><td>Beckman Coulter</td><td>344057</td><td></td></tr><tr><td>Conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Gradient maker</td><td>BioComp Gradient Master</td><td>see Anal Biochem. 1985 Jul;148(1):254-9.</td><td></td></tr><tr><td>Syringe and Blunt Cannula</td><td>Monoject</td><td>8881513918 and 888202017</td><td></td></tr><tr><td>Wide-bore pipette tips</td><td>Fischerbrand</td><td>02-707-134</td><td></td></tr><tr><td>Plaque counter</td><td>New Brunswik Scientific</td><td>Colony Counter Model C-110</td><td></td></tr><tr><td>Culture tubes</td><td>Fischerbrand</td><td>14-961-33</td><td></td></tr><tr><td>Cell-free system</td><td>Mycroarray Inc</td><td>Mytxtl</td><td></td></tr><tr><td>BioComp Gradient Master</td><td>BioComp Instruments</td><td>Model 105ME</td><td></td></tr><tr><td>LB agar plate recipe</td><td></td><td></td><td>25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td></tr></tbody></table>

synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

試験管反応の基礎および応用科学を実行する新しい機能を提供するより汎用性とモジュール性、エンジニア リング、無細胞転写・翻訳 (TXTL) システムの新世代。過去 10 年間携帯無料 TXTL 斬新な学際的な研究領域定量と合成に関連する生物学の広い範囲のための強力な技術となっています。新しい TXTL プラットフォームは、構築し、合成又は天然の遺伝子回路の実施により生化学システムを尋問すると特に便利です。体外TXTL 実証されています急速にプロトタイプ規制要素と生物学的ネットワーク同様に要約として便利な分子の自己組織化メカニズムは、生活システム。この資料でどのように伝染性のバクテリオファージ、MS2 (RNA)、ΦΧ174 などを述べる (ssDNA) および T7 (dsDNA) 完全すべて大腸菌、無細胞 TXTL 系を用いたワンポット反応で彼らのゲノムから合成されます。3 つの coliphages の合成は、プラクの試金を使用して定量化されます。どのように合成バクテリオファージの収量反応の生化学的設定に依存を示します。ペグの 8000 系の制御された濃度をエミュレート分子クラウディング大きさの命令によって合成されたファージの量に影響します。バクテリオファージを増幅する方法と彼らのゲノムを浄化する方法について述べる。プロトコルとこの作品で示された結果のセットは無細胞合成生物学と工学の学際的な研究者に関心のはずです。

4 つの代表的な結果を示します。図 1、携帯無料 TXTL システムとファージ DNA 株式はリビングで汚染されないように否定的なコントロールのセットを提案するエシェリヒア属大腸菌のセル。我々 はそのまま大腸菌の無細胞 TXTL システムが無いことを確認 (図 1A 図 1B</strong...

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

大腸菌の伝染性のバクテリオファージの合成による無細胞発現系

無細胞転写・翻訳プラットフォームの新世代は、生化学システムの in vitro遺伝子回路の実行を構築するために設計されています。この記事で私たちはすべて大腸菌無細胞 TXTL システムを使用して彼らのゲノムからのバクテリオファージ MS2、ΦΧ174、T7 などの合成方法について説明します。

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

14.5K ビュー.  University of Minnesota. 無細胞転写・翻訳プラットフォームの新世代は、生化学システムの in vitro遺伝子回路の実行を構築するために設計されています。この記事で私たちはすべて大腸菌無細胞 TXTL システムを使用して彼らのゲノムからのバクテリオファージ MS2、ΦΧ174、T7 などの合成方法について説明します。

ビデオ: Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

免疫・感染学

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

生物学

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.

大腸菌の無細胞タンパク質合成のベース: 堅牢な柔軟性とアクセス可能なプラットフォーム技術のためのプロトコル

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

<meta charset="utf8"></head><body>マイクロコンパートメント化合成細胞のためのCFPSプロトコル

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.

<meta charset="utf8"></head><body>著者スポットライト:合成細胞構築のためのCFPSシステムの最適化

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.

合成細胞を構築するための無細胞タンパク質合成システム

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 ...

生化学

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.

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

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

生物工学

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.

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

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

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.

無細胞自己誘導ワークフローを使用した24時間以内に細胞から無細胞タンパク質合成へ

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

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

実装するためのプロトコル<em&gt;エシェリヒア·コリ</em合成生物学のための&gt;に基づくTX-TLの無細胞発現系

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

大腸菌の伝染性のバクテリオファージの合成による無細胞発現系

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大規模な挿入環境ゲノムライブラリーの製造

大腸菌の無細胞タンパク質合成のベース: 堅牢な柔軟性とアクセス可能なプラットフォーム技術のためのプロトコル

合成細胞を構築するための無細胞タンパク質合成システム

合成細胞を構築するための無細胞タンパク質合成システム

合成細胞を構築するための無細胞タンパク質合成システム

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

無細胞自己誘導ワークフローを使用した24時間以内に細胞から無細胞タンパク質合成へ

実装するためのプロトコル