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.).

<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>

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><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

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

Universidad de Cartagena UDEC

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

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.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

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

Forschung

Immunologie und Infektion

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.

Large Insert Environmental Genomic Bibliothek Produktion

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

Biologie

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.

Escherichia coli-basierte zellfreie Proteinsynthese: Protokolle für eine robuste, flexible und zugängliche Plattform-Technologie

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

Chemie

<meta charset="utf8"></head><body>CFPS-Protokolle für mikrokompartimentierte synthetische Zellen

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>Autor im Rampenlicht: Optimierung von CFPS-Systemen für den Bau synthetischer Zellen

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.

Zellfreies Proteinsynthesesystem für den Aufbau synthetischer Zellen

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 ...

Biochemie

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.

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

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

Biotechnik

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.

Herstellung von Proteinproduktion synthetischer Zellen mit zellfreien bakteriellen Extrakten, Liposomen und Emulsionstransfer

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.

Von der Zelle zur zellfreien Proteinsynthese innerhalb von 24 Stunden mit zellfreiem Autoinduktions-Workflow

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.

Protokolle für die Implementierung einer<em&gt; Escherichia coli</em&gt; Basierend TX-TL Zellfreie Expression System für Synthetische Biologie

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

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

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Zellfreies Proteinsynthesesystem für den Aufbau synthetischer Zellen

Zellfreies Proteinsynthesesystem für den Aufbau synthetischer Zellen

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Herstellung von Proteinproduktion synthetischer Zellen mit zellfreien bakteriellen Extrakten, Liposomen und Emulsionstransfer

Von der Zelle zur zellfreien Proteinsynthese innerhalb von 24 Stunden mit zellfreiem Autoinduktions-Workflow

Protokolle für die Implementierung einer