図1、2、3、4、6、8及び表2、3、10、15、16は、参考文献29から変更されています。この作品は、国立科学財団（NSF-GSにCCF 1117143およびNSF-CCF 1162141を付与する）によってサポートされていました。 Y.-JCは、台湾政府のフェローシップによってサポートされていました。 SDRは、国立科学財団大学院研究フェローシッププログラム（GRFP）によってサポートされていました。

<ol>
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The authors declare no competing financial interests.

本論文では、高純度のプラスミドDNAからndsDNAゲートを導出するための方法を説明します。また、プロトコルは、蛍光動力学アッセイを用いて、ゲート性能を特徴付けるために提示されています。実験データは、プラスミド由来の系、合成系は、ポリアクリルアミドゲル電気泳動（PAGE）を用いて精製したストランドから組み立てられていても、その合成対応物よりも優れていることを示しています。多分、プラスミド由来のゲートの性能の改善は、主に生物学的DNAの非常に高純度に起因するものです。合成DNAは、さまざまなエラーが含まれ、特に、長さN-1のオリゴヌクレオチド、およびそのような副生成物を生じる欠失は、典型的には、完全PAGEまたは高速液体クロマトグラフィー（HPLC）精製手順で除去されません。ここで報告されたものと同様の改善はまた、生物学的供給源21に由来するDNAを使用した触媒ヘアピン増幅器の以前の研究で観察されました。 <p clお尻= &quot;jove_content&quot;&gt;しかし、プラスミド由来のゲートを用いても、完全に少なくとも2つの理由がありますするためのゲート性能のエラー、排除することはできません。あまりにも多くてゲートにつながることができ、最初の過消化またはカット精度の欠如を間違った位置にニックまたはニック。いずれの場合においても、ゲートが望ましくない反応に関与する可能性が高いです。このような問題は、（ 図4を参照）を使用する酵素の量を最適化することにより緩和することができます。第二に、これらの実験では、ほとんどの入力および補助鎖合成DNAであり、したがって、欠失および変異を含みました。原則として、すべての一本鎖入力と補助鎖はまた、事前符号化M13ウイルスゲノム26のニッキング酵素消化を介してファージミドDNAから得ることができます。おそらく回路性能は、さらに、細菌ゲノムに由来​​する一本鎖DNAを用いて改善することができます。 プラスミド由来のゲートの使用は、回路性能を改善することが見出されたが、分析コストや処理時間のプラスミド由来のゲートの製造がやや安い（ 表15）であるが、それは商業的に合成されたオリゴ（ 表16）からゲートのアセンブリおよび精製に比べて2~3倍長い処理時間を要することが明らかになりました。プラスミド由来のゲートの主要コストは、遺伝子合成、制限酵素の使用です。 （30 nmで15の反応のために十分な）ゲートの300ピコモルのために、参加ゲートの推定コストはフォークゲート、違いによるニッキング酵素の使用にいるコストの差は約$ 170と$ 200です。これとは対照的に、PAGE精製料込み$ 260の周りに同じゲー​​ト費用鎖の化学合成。プラスミド由来のゲートのための主要な時間コストは、単にDNA合成と同様に、遺伝子合成会社に委託することができ、クローニング手順、です。しかし、一旦組み立て、プラスミド由来のゲートは、宿主プラスミドを容易に複製することができるという利点を有しますND細菌のグリセロールストックの形で保存することができます。これは、上にゲートを何度も再利用することができます。 楽しみにして、プラスミド由来のゲートの改良された性能を実験的にDNA CRNsで、これまでに実証されているよりも、力学挙動のはるかに大きな範囲を可能にすることができます。例えば、最近の理論的研究47,48は、マクロスケールでの自己組織化の空間パターンは、反応拡散機構を介してDNA CRNsで実現することができることを示唆しました。ここで紹介する方法は、自己パターニングDNA材料の基礎となる分子成分を構築するための実行可能なパスを提供します。挑戦が、プログラム可能な方法で、マクロスケールの形態を開発するバイオマテリアルの研究から再生医療に至るまでの分野で重要な意味を持っているでしょう。

ワトソン-クリック塩基対の予測可能性は、動的なDNAのナノテクノロジーは、動的特性1,2と分子デバイスを設計するためのプログラム可能な方法として出現することができました。特に、DNA鎖置換 - プログラム可能な、競合ハイブリダイゼーション反応は - 動的DNAシステムを操作するための強力な機構であることが判明しました。 DNAの鎖置換反応では、入ってくるオリゴヌクレオチドは、相補的結合パートナーか​​らの以前に結合した「出力」鎖を置換します。複数のそのような反応は、個々の反応3ステップの順序やタイミングを制御度の高い多段階反応カスケードに一緒に連鎖させることができます。 DNA鎖変位カスケードは4-7、切り替え可能なナノ構造体8-10、自律分子モーター11-15、および非共有結合触媒アンプ13,16-21デジタルとアナログの分子回路を生成するために使用されています。また、D鎖置換反応を用いてNAデバイスをシミュレートし、コンピュータ支援設計ソフトウェア22-24を使用して 、多様な用途のために設計することができます。 現在、化学的に合成されたDNAは、DNAナノテクノロジーのための主材料として機能します。しかし、DNA合成プロセスにおけるエラー、そして得られた不完全なオリゴヌクレオチドは、誤った副反応を引き起こすことにより、動的DNAデバイスの性能を制限すると考えられています。例えば、「リーク」反応も反応トリガーの非存在下での出力オリゴヌクレオチドの放出をもたらすことができます。このような効果は、最初のリークであっても最小限の量は、最終的にカスケード19,20の完全な活性化につながる自己触媒反応のカスケードに最も明白です。一部のコンポーネントが意図した入力7,25の存在下でさえも誘発しないので逆に、反応はしばしば活性化の期待されるレベルに到達することができません。 DNAベースの性能を作成するにはそれらの生物学的なタンパク質ベースの対応物に匹敵するナノデバイスは、エラーモードが劇的に低減する必要があります。 細菌プラスミドまたは他の生物学的DNAは、ナノテクノロジーのアプリケーションのための高純度のDNAの比較的安価な供給源として働く可能性があります。 DNAの大量の細菌中で複製することにより生成することができ、生物系の固有の校正能力は、得られたDNAの純度を確保します。実際には、いくつかの最近の論文は、ナノテクノロジーのアプリケーション21,26-28のための生物学的DNAの潜在的な有用性を認識しています。しかしながら、プラスミドDNAの完全に二本鎖の性質は、これまで一般的に複数のオリゴヌクレオチドからなり、二本鎖および一本鎖ドメインの両方を含む動的DNAデバイスを製造するための材料としての使用を禁止しています。最近の論文29この問題が対処された、主にニックの入った二本鎖DNA（ndsDNA）で構成されて新たなDNAのゲートアーキテクチャでは紹介しましたD。 重要なことには、ndsDNAゲートのシステムは、正式な化学反応ネットワーク（CRN）29で指定されたダイナミクスを実現するように設計することができます。 ndsDNAゲートは、このように振動とカオス、双安定性とメモリ、ブール論理またはアルゴリズムの挙動30-38を示 ​​す動的システムを作成するには、原則として、使用することができます。例えば、参考文献29は、「コンセンサス」プロトコル、分散コンピューティングアルゴリズム29,39,40のタイプの分子の実装を提供三反応CRNを示しました。この作品は、最初に急速に機能性分子システムを合成するための「プログラミング言語」（ 図1A）などのCRNの形式化のための新規な使用を実証しました。 ここでは、プラスミドDNAからndsDNAゲートを導出するための詳細なプロトコルが提供されます。最初のシーケンス設計プロセスのレビューです。そして、どのように含む合成オリゴヌクレオチドの説明を次のゲート配列は、細菌培養物を介して検証され、増幅されたプラスミドとの配列中にクローニングされます。 ndsDNAゲートは、酵素処理によりプラスミドから誘導することができる方法を次に、それが示されている（ 図2参照）。最後に、蛍光動力学アッセイを用いてゲートの動作をテストするための方法を概説します。 反応機構 例として、プロトコルは、触媒化学反応A + B-&gt; B + Cに焦点を当てています。種A、B、およびC（「シグナル」、 図1B）は、すべての異なる一本鎖DNA分子に対応します。これらの分子の配列は完全に独立しており、鎖は互いに直接反応しません。すべての信号のシーケンス、すなわち二つの異なる機能的ドメイン、鎖置換反応において一緒に作用する部分配列を有する：1）短い足掛かりドメイン（ラベル鎖置換rの開始のために使用され、TB、TC）TA信号の同一性を決定するeaction、及び2）長いドメイン（A、B、Cのラベル）。 信号鎖間の相互作用は、ニックの入った二本鎖DNA（ndsDNA）ゲート複合体（ABとフォークBCに参加呼ばれる）と補助一本鎖種（&lt;TR R&gt;、&lt;BのTR&gt;、&lt;CのTB&gt;と&lt;私のTCによって媒介されます&gt;）。正式な反応A + B-&gt; B + Cは、各反応工程は、その後の反応（ 図1B）のための足掛かりを公開鎖置換反応の一連のステップを介して実行されます。信号Cは、フォークゲートに結合している間に、この例では、信号A及びBは、最初に溶液中で遊離しています。反応BとCの終了時に溶液中にあります。より一般的には、ゲートにバインドされた信号は、溶液中で遊離している信号がアクティブな間に、つまり、彼らは鎖置換反応などに参加することができ、非アクティブです入力。反応の時間経過は、蛍光レポーター戦略（ 図1C）を使用して続いています。以前の研究29には、この反応機構は、正しい化学量論だけでなく、目的とする反応の速度を実現するだけでなく、ことが示されました。

<table border="0">
	<tbody>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>NEB</td>
			<td>M0531S</td>
			<td></td>
		</tr>
		<tr>
			<td>PvuII-HF</td>
			<td>NEB</td>
			<td>R3151L</td>
			<td></td>
		</tr>
		<tr>
			<td>PstI-HF</td>
			<td>NEB</td>
			<td>R3140S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth, Modified</td>
			<td>SIGMA-ALDRICH</td>
			<td>T0918-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Hispeed Maxi-prep Kit</td>
			<td>QIAGEN</td>
			<td>12662</td>
			<td></td>
		</tr>
		<tr>
			<td>Nb.BsrDI</td>
			<td>NEB</td>
			<td>R0648L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nt.BstNBI</td>
			<td>NEB</td>
			<td>R0607L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-stranded Genomic Blocks</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horiba Jobin-Yvon Spex Fluorolog-3 Fluorimeter</td>
			<td>Horiba/Jobin Yvon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Quartz Cells&#160;</td>
			<td>Starna</td>
			<td>23-5.45-S0G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Backbones</td>
			<td>BioBrick</td>
			<td>E0240-pSB1A2</td>
			<td>High copy number plasmid with Ampicillin resistance. Sequence can be found from http://parts.igem.org</td>
		</tr>
	</tbody>
</table>

plasmid-derived dna strand displacement gates for implementing chemical reaction networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

実装化学反応ネットワーク用プラスミド由来のDNA鎖置換ゲイツ

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

plasmid-derived-dna-strand-displacement-gates-for-implementing

Research

JoVE Journal

Biology

14.0K ビュー.  University of Washington. This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. 

ビデオ: Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

哺乳動物細胞におけるDNA二重鎖切断（DSB）修復の解析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

生物学

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

どのように文化、レコードとマイクロ電極アレイで神経ネットワークを刺激する（多国間環境協定）へ

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

免疫不全マウスにおける生体工学ヒト微小血管ネットワーク

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

SeqA-DNA複合体の結晶化のためのDNA二重鎖の反復最適化

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

ジェネラリストの腸内で代謝的に活性な細菌の同定<em&gt;ハスモンヨトウリットラリス</em&gt; DNAを安定同位体を経由して使用するプロービング<sup&gt; 13</sup&gt; C-グルコース

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

プロトタイプ小型化フローサイトメーターおよびコンパニオンマイクロ流体混合技術の縮小重力環境ハードウェアデモンストレーション

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

ラットの肝臓におけるDNA鎖切断および酸化的DNA損傷を測定するためのインビボアルカリコメットアッセイおよび酵素修飾されたアルカリコメットアッセイで

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...