Dieses Material basiert auf Arbeit unterstützt durch das Office of Naval Research Award Nummer N00014-13-1-0074 (v.n.), das Human Frontier Science Program gewähren, Anzahl RGP0037/2015 (v.n.), und der binationalen Wissenschaftsstiftung gewähren 2014400 (v.n.).

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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. 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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. 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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>

Die Autoren erklären die folgenden konkurrierenden finanziellen vorgibt: Noireaux Labor erhält Forschungsgelder von MYcroarray, ein Händler von MYtxtl zellfreien Protein Ausdruck Kit.

Nach der Technik in Chen, Et Al. 14 für SPMC ist ein wichtiger Schritt erreicht, wenn die richtigen Bedingungen für Superinfektion zu bestimmen. Der Parameter, der am ehesten ein Host Stamm Fähigkeit steuert, Superinfektion zu widerstehen ist häufig der Ausgangskonzentration des infizierenden Phagen. Die Wirtszellen muss logarithmischen Wachstumsphase vor Erstinfektion mit einer sehr kleinen Menge von Phagen. Schließlich die Phagen werden auch logarithmische Wachstum erreichen und da...

Im letzten Jahrzehnt wurde die zellfreie Expression Technologie zur Adresse neuartige Anwendungen im aufstrebenden multidisziplinäre Forschung Bereiche im Zusammenhang mit synthetischen und quantitative Biologie entwickelt. Ursprünglich verwendet, um Proteine, die unabhängig von einem lebenden Organismus auszudrücken, wurden zellfreie TXTL Neuanlagen für beide Grundlagenforschung und der angewandten Wissenschaften1,2, Erweiterung des Geltungsbereichs dieser Technologie deutlich entwickelt. Die neue Generation der TXTL Plattformen wurde entwickelt, um benutzerfreundlich, werden effizienter (bis 2 mg/mL der Proteinsynthese im Batch-Modus3), mehr auf der Ebene der Transkription4vielseitig und modulare damit problemlos integrieren Roman natürliche oder synthetische Funktionen, die die Möglichkeiten von vorhandenen biologischen Systemen5,6. Insbesondere zellfreie TXTL Systeme für das rapid Prototyping von genetischen Programmen, wie regulatorische Elemente oder kleine genetische Schaltkreise7,8,9, handlich geworden durch die Reduzierung der Design-Build-test Radeln Sie nach ein paar Tagen. Bemerkenswert ist, sind die neuen TXTL-Systeme verarbeiten große DNA-Programme wie die komplette Synthese von Coliphages10,11, demonstrieren stark genug Leistung um die Rekonstitution der genomischen aktiv unterstützen Lebewesen DNA kodiert.
TXTL Systeme präsentieren viele technische Vorteile gegenüber traditionellen in-vitro- konstruktive biochemischen Assays. Zellfreie TXTL verbindet den Prozess der Genexpression bis zum fertigen Produkt in einem reduzierten und offenen Umfeld, im Gegensatz zu den komplexen Zytoplasma einer lebenden Zelle. TXTL verwendet DNA biochemischen Systemen in Vitrozu rekonstruieren, die ist mit modernen DNA Montagetechniken, bezahlbar und schnell neben nicht erfordern anspruchsvolle Protein Reinigungsschritte. Zellfreie Ausdruck bietet direkten Zugriff auf die meisten Komponenten in den biochemischen Reaktionen, wodurch eine tiefere Dissektion der molekularen Wechselwirkungen12. In einer TXTL Reaktion kann man die biochemischen und biophysikalischen Einstellungen nach Belieben, ändern, fast unmöglich in einer lebenden Zelle. Angesichts dieser Vorteile und Verbesserungen, wird die TXTL-Technologie mehr und mehr populär als eine alternative Plattform für synthetische und quantitative Biologie. Während die Forschungsgemeinschaft mit TXTL rasant wächst und TXTL eine Standardtechnologie in Bioingenieurwesen wird, es ist wichtig zu verstehen, wie solche Plattformen um angemessene Praktiken im Zusammenhang mit der Ausführung der TXTL Reaktionen und zu entwickeln die Interpretation der Ergebnisse.
In diesem Artikel beschreiben wir wie ein alle E. Coli TXTL System verwenden, um in ein-Topf-Reaktionen, Bakteriophagen von ihrem Genom11, z. B. MS2 zu synthetisieren (RNA, 3,4 kb), ΦΧ174 (SsDNA, 5,4 kb), und T7 (DsDNA, 40 kb). Wir zeigen, wie die Höhe der Phagen Änderungen in Bezug auf einige der biochemischen Einstellungen der Reaktionen (Magnesium und Kalium-Konzentrationen) synthetisiert. Molekulare drängen, durch eine Reihe von PEG 8000 Konzentrationen emuliert hat einen enormen Einfluss auf die Phagen Synthese über mehrere Größenordnungen. Die Realisierung solcher großen biochemische Systeme im einzelnen Reagenzglas Reaktionen, die gleichzeitig die Prozesse der Transkription, Übersetzung, rekapitulieren und Selbstmontage, ist interessant für grundlegende Fragen im Zusammenhang mit Biologie und Biophysik 10 (Genregulation, Self-assembly), sowie für die Entwicklung von Anwendungen, wie z. B. Umnutzung Phagen-Funktionen, um neue Nanostrukturen13bauen. Neben einem praktischen auf TXTL bieten wir Methoden für Phagen Verstärkung, Genom-Extraktion und Reinigung und Phagen Quantifizierung von Plaque-Assay. In diesem Manuskript vorgestellten Methoden eignen sich für Forscher, die mit E. Coli Extrakt basiert zellfreien Systemen und interessieren sich für Bakteriophagen.
In dieser Arbeit vorgestellten Protokolle können wie folgt zusammengefasst werden: (1) Phagen Verstärkung (Tag1: Inokulation vorzubereiten Zellen, Tag 2: einzelne Plaque, mehrere Phagen Wachstum und Konzentration und Tag 3: Reinigung des Phagen), 2) Double-Stranded Genom DNA-Extraktion (Phenol/Chloroform Extraktion), und 3) zellfreie Phagen Reaktion und Titer Experiment (Tag1: Platte Wirtszellen und machen Agarplatten, Tag 2: zellfreie Reaktion und Host cell Vorkultur und Tag 3: Host Zelle Kultur und Phage Titer).

<table width="899">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:168pt" width="224" />
		<col style="width:219pt" width="292" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:162pt;height:15.75pt" width="216">Ultracentrifugation tubes</td>
			<td class="xl21" style="width:168pt" width="224">Beckman Coulter</td>
			<td class="xl21" style="width:219pt" width="292">344057</td>
			<td class="xl20" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Conical tubes</td>
			<td class="xl22" style="width:168pt" width="224">Falcon</td>
			<td class="xl21">352070</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Gradient maker</td>
			<td class="xl21">BioComp Gradient Master</td>
			<td class="xl22" style="width:219pt" width="292">see Anal Biochem. 1985 Jul;148(1):254-9.</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Syringe and Blunt Cannula</td>
			<td class="xl21">Monoject</td>
			<td class="xl21">8881513918 and 888202017</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Wide-bore pipette tips</td>
			<td class="xl21">Fischerbrand</td>
			<td class="xl21">02-707-134</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Plaque counter</td>
			<td class="xl21">New Brunswik Scientific</td>
			<td class="xl21">Colony Counter Model C-110</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Culture tubes</td>
			<td class="xl22" style="width:168pt" width="224">Fischerbrand</td>
			<td class="xl21">14-961-33</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Cell-free system</td>
			<td class="xl22" style="width:168pt" width="224">Mycroarray Inc</td>
			<td class="xl22" style="width:219pt" width="292">Mytxtl</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">BioComp Gradient Master</td>
			<td class="xl17" style="width:168pt" width="224">BioComp Instruments</td>
			<td class="xl17" style="width:219pt" width="292">Model 105ME</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">LB agar plate recipe</td>
			<td class="xl20"></td>
			<td class="xl20"></td>
			<td class="xl24" style="width:512pt" width="683">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

Eine neue Generation von zellfreien Transkription-Übersetzung (TXTL) Systeme, entwickelt, um eine größere Vielseitigkeit und Modularität, neuartige Funktionen zur Grundlagenforschung und der angewandte Wissenschaften in Reagenzglas Reaktionen führen zu bieten haben. Im letzten Jahrzehnt ist die zellfreie TXTL eine leistungsfähige Technik für eine Vielzahl von neuartigen multidisziplinäre Forschung Bereiche im Zusammenhang mit quantitativen und synthetische Biologie geworden. Die neue TXTL-Plattformen sind besonders nützlich, um zu konstruieren und biochemische Systemen durch die Ausführung von synthetischen oder natürlichen gen Schaltungen zu verhören. In-vitro- TXTL nachweislich schnell Prototyp regulatorische Elemente und biologischer Netzwerke sowie Zusammenfassend bequem molekulare Mechanismen Selbstmontage in lebenden Systemen gefunden. In diesem Artikel beschreiben wir wie infektiöse Bakteriophagen, z. B. MS2 (RNA), ΦΧ174 (SsDNA) und T7 (DsDNA), sind völlig von ihrem Genom im ein-Topf-Reaktionen mit allen Escherichia coli, zellfreie TXTL System synthetisiert. Synthese von den drei Coliphages wird mit dem Plaque-Assay quantifiziert. Wir zeigen, wie die Rendite der synthetisierten Phagen biochemische Reaktionen-Einstellungen abhängig ist. Molekulare Verdrängung durch eine kontrollierte Konzentration von PEG 8000 emuliert beeinflusst die Höhe des synthetisierten Phagen durch Aufträge von Größen. Wir beschreiben auch die Phagen zu verstärken und ihre Genome zu reinigen. Protokolle und Ergebnisse, die in dieser Arbeit sollten in zellfreien synthetische Biologie und Biotechnologie an multidisziplinären Forscher beteiligt werden.

Wir zeigen vier repräsentative Ergebnisse. In Abbildung 1, präsentieren wir eine Reihe von negativen Kontrollen sicherstellen, dass das zellfreie TXTL-System und die Phagen-DNA-Bestände nicht mit lebenden kontaminiert sind E. Coli Zellen. Wir überprüfen, dass das zellfreie TXTL System intakt E. Coli ist Zell-Kontamination durch Ausplattieren beide ein nicht inkubiert und inkubierten Reaktionslösung leer von genomischer DNA (<strong cla...

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

Synthese von infektiösen Bakteriophagen in einem E. Coli -basierte zellfreie Expressionssystems

Eine neue Generation von zellfreien Transkription-Übersetzung Plattformen wurde entwickelt, um die biochemischen Systemen in Vitro durch die Ausführung des Gens Schaltungen aufbauen. In diesem Artikel beschreiben wir, wie aus ihrem Genom mit einem alle E. Coli zellfreie TXTL System Bakteriophagen MS2, ΦΧ174 und T7, synthetisiert werden.

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

Research

JoVE Journal

Bioengineering

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

14.5K Aufrufe.  University of Minnesota. Eine neue Generation von zellfreien Transkription-Übersetzung Plattformen wurde entwickelt, um die biochemischen Systemen in Vitro durch die Ausführung des Gens Schaltungen aufbauen. In diesem Artikel beschreiben wir, wie aus ihrem Genom mit einem alle E. Coli zellfreie TXTL System Bakteriophagen MS2, ΦΧ174 und T7, synthetisiert werden.

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

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bakterielle Erkennung und Identifizierung mit elektrochemischen Sensoren

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Forschung

Biotechnik

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Synthese einer Intein-vermittelten Protein-Hydrogel Künstliche

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Eine neuartige Operationstechnik als Grundlage für In Vivo teilweise Leber Engineering in der Ratte

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

Zellfreien Protein Ausdruck mithilfe der rasch wachsenden Bakterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

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.

Eine multilayer mikrofluidische Plattform für die Leitung einer längeren zellfreien Genexpression

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

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

High-Throughput Identifikation der Resistenz gegen Pseudomonas syringae pv. Tomate in Tomaten mit Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Ein In-vitro-System zur Messung der thrombolytischen Wirksamkeit von Histotripsie und einem lytischen Medikament

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

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.

OnePot PURE Zellfreies System

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

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.

Schnelle Charakterisierung genetischer Teile mit zellfreien Systemen

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.