Diese Arbeit wurde durch das LRIR 16RH3003J-Stipendium des Air Force Office of Scientific Research sowie durch das Programm Synthetic Biology for Military Environments Applied Research for the Advancement of S&T Priorities (ARAP) des U.S. Office of the Under Secretary of Defense for Research and Engineering unterstützt.
Die Plasmidvektorsequenz für ThyRS (pSALv-RS-GFPa1, 3,4 kb) wurde großzügigerweise von Dr. J. Gallivan zur Verfügung gestellt. Seidenraupenkokons von Bombyx mori wurden großzügig von Dr. D.L. Kaplan von der Tufts University, MA, gespendet.

<ol>
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N., 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=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6 (10), 1612-1631 (2011).</li><li>Drachuk, I., 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=Silk+macromolecules+with+amino+acid-Poly(Ethylene+Glycol)+grafts+for+controlling+layer-by-layer+encapsulation+and+aggregation+of+recombinant+bacterial+cells.">Silk macromolecules with amino acid-Poly(Ethylene Glycol) grafts for controlling layer-by-layer encapsulation and aggregation of recombinant bacterial cells.</a> ACS Nano. 9 (2), 1219-1235 (2015).</li><li>Antipov, A. A., Sukhorukov, G. 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Die hier dargestellten Ansichten und Meinungen sind die der Autoren und repräsentieren nicht unbedingt die Ansichten des DoD oder seiner Komponenten

Selektiv permeable Hydrogel-Mikrokapseln, die mit verschiedenen Arten von DNA-kodierten Sensordesigns beladen sind, können nach diesem Protokoll hergestellt werden. Eine der Besonderheiten des LbL-Ansatzes ist die Fähigkeit, die Komplexität von Mikrokapseln während der Bottom-up-Assemblierung maßzuschneidern, die in der Regel mit der Adsorption molekularer Spezies an Opfervorlagen beginnt. Durch sorgfältige Anpassung der Konzentrationen der Ausgangskomponenten, der pH-Bedingungen und der Anzahl der Schichten könne...

Das Gebiet der synthetischen Biologie bietet einzigartige Möglichkeiten, Sensorfähigkeiten zu entwickeln, indem natürliche Mechanismen genutzt werden, die von Mikroorganismen entwickelt wurden, um ihre Umwelt und potenzielle Bedrohungen zu überwachen. Wichtig ist, dass diese Sensormechanismen in der Regel mit einer Reaktion verbunden sind, die diese Mikroorganismen vor schädlicher Exposition schützt und die Genexpression reguliert, um negative Auswirkungen zu mildern oder die Aufnahme giftiger Materialien zu verhindern. Es wurden erhebliche Anstrengungen unternommen, um diese Mikroorganismen so zu verändern, dass sie Ganzzellsensoren herstellen, die diese natürlichen Reaktionen nutzen, sie aber umleiten, um neue Ziele zu erkennen und/oder ein messbares Signal zu erzeugen, das zu Quantifizierungszwecken gemessen werden kann (typischerweise Fluoreszenz)1,2. Gegenwärtig deuten Bedenken hinsichtlich der Verwendung genetisch veränderter Mikroorganismen (GVO), insbesondere bei der Freisetzung in der Umwelt oder im menschlichen Körper, aufgrund des Austritts ganzer Zellen oder eines Teils ihres genetischen Materials, selbst wenn es in einer Polymermatrix eingekapselt ist, darauf hin, dass alternative Wege zur Nutzung dieser Sensoransätze erforderlich sind3.
Ein wirksamer Ansatz, um die Vorteile der mikroorganismenbasierten Sensorik zu nutzen, ohne sich Gedanken über den Einsatz von GVO machen zu müssen, ist die Verwendung von In-vitro-Transkriptions-/Translationssystemen (IVTT). Aus praktischer Sicht bestehen IVTT-Systeme aus einer Mischung, die die meisten Zellbestandteile in einem aktiven Zustand enthält, die auf unterschiedliche Weise aus den Zellen "extrahiert" wurden,z. B. durch Beschallung, Perlenschlagen oder andere 4. Das Endprodukt dieses Prozesses ist ein biochemisches Reaktionsgemisch, das bereits für die Transkription und Translation optimiert ist und zum Testen verschiedener Sensoren in einem "offenen Gefäß"-Format verwendet werden kann, ohne die Einschränkungen, die mit der Verwendung ganzer Zellen verbunden sind (Membrandiffusion, Transformationseffizienz, Zelltoxizität usw.). Wichtig ist, dass verschiedene Sensorkomponenten quantitativ hinzugefügt und ihre Wirkung durch verschiedene optische und spektrometrische Techniken untersucht werden können, wie wir gezeigt haben5. Es wurde festgestellt, dass die Leistung von IVTT-Systemen inkonsistent sein kann. Neuere Studien haben jedoch Ansätze zur Standardisierung ihrer Präparation und Charakterisierung gezeigt, was bei der Untersuchung ihrer Leistung im Sensordesign eine große Hilfe ist6. In jüngster Zeit wurden viele Beispiele für IVTT-Systeme demonstriert, die zur Herstellung papierbasierter Assays durch die Gefriertrocknung ihrer Komponenten in Papiermatrizes verwendet werden, einschließlich der Detektion von Schwermetallionen, Drogen, Quorum-Sensorelementen und anderen 7,8,9. Ein spannender Anwendungsbereich für IVTT-basierte Sensoren ist ihr Einsatz in Sensoranwendungen in verschiedenen Arten von Umgebungen, einschließlich Boden, Wasser und dem menschlichen Körper. Um diese IVTT-Systeme in diesen anspruchsvollen Umgebungen einsetzen zu können, muss ein Kapselungsansatz implementiert werden, um die IVTT-Komponenten einzudämmen und sie vor Verschlechterung zu schützen.
Die gebräuchlichsten Verkapselungsansätze für IVTT-Systeme umfassen die Verwendung von Lipidkapseln, Mizellen, Polymersomen und anderen dicht geschlossenen Mikrobehältern10,11,12. Ein Nachteil dieses Ansatzes ist die Notwendigkeit, entweder passive oder aktive Mechanismen für den Transport von Materialien in und aus den Behältern zu integrieren, um die Kommunikation mit der externen Umgebung zu ermöglichen und Sensorfunktionen bereitzustellen. Um einige dieser Probleme zu überwinden, berichtet die Studie hier über eine Methode, die einen einfachen, aber effektiven Ansatz zur Verkapselung der Kodierungsmaterialien für verschiedene Sensordesigns bietet, die in IVTT-Systemen ausgedrückt werden sollen. Dieser Ansatz basiert auf der Verwendung der Layer-by-Layer (LbL)-Abscheidung eines Biopolymers in Gegenwart der interessierenden Plasmide, um hohle Mikrokapseln mit hoher Porosität herzustellen, die es dem geschützten genetischen Material ermöglichen, mit den verschiedenen Komponenten des IVTT der Wahl zu interagieren. Die Studie zeigte, dass verkapselte Plasmide die Transkription und Translation steuern können, wenn sie innerhalb dieser polymeren Matrix aktiviert werden, wie die Reaktion eines Plasmid-kodierten Aptamers und eines Riboschalters auf ihre entsprechenden Ziele zeigt. Zusätzlich schützt diese LbL-Beschichtung die Plasmide monatelang ohne besondere Lagerbedingungen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T)</td>
			<td>Lucerna</td>
			<td>DFHBI-1T</td>
			<td></td>
		</tr>
		<tr>
			<td>5x T4 DNA Ligase Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>46300-018</td>
			<td></td>
		</tr>
		<tr>
			<td>6x Blue Gel Loading Dye</td>
			<td>New England BioLabs</td>
			<td>B7021S</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, black circular</td>
			<td>Corning</td>
			<td>3601</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>BioReagent, for molecular biology, low EEO</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>powder or crystals, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>BlpI restriction enzymes</td>
			<td>New England BioLabs</td>
			<td>R0585S</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Disposable Vacuum Filter/Storage Systems</td>
			<td>FisherScientific</td>
			<td>09-761-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide, DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td>ACS reagent, &#8805;99.9%</td>
		</tr>
		<tr>
			<td>DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt.</td>
			<td>Addgene</td>
			<td>Plasmid #66788</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLightTM550 Antibody Labeling kit (Invitrogen)</td>
			<td>ThermoFisher Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli S30 extract system for circular DNA</td>
			<td>Promega</td>
			<td>L1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Conical centrifuge tubes, 15 mL</td>
			<td>FisherScientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Conical centrifuge tubes, 50 mL</td>
			<td></td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Microcentrifuge tubes, 1.5 mL</td>
			<td>FisherScientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrofluoric acid, HF</td>
			<td>Sigma-Aldrich</td>
			<td>695068</td>
			<td>ACS reagent, 48%</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td>mixture of Kanamycin A (main component) and Kanamycin B and C</td>
		</tr>
		<tr>
			<td>KpnI restriction enzymes</td>
			<td>New England BioLabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar plate supplemented with 100 &#181;g/mL ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>L5667</td>
			<td>pre-poured agar plates with 100 &#181;g/mL ampicillin</td>
		</tr>
		<tr>
			<td>LB agar plate supplemented with 50 &#181;g/mL kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>L0543</td>
			<td>pre-poured agar plates with 50 &#181;g/mL kanamycin</td>
		</tr>
		<tr>
			<td>LB broth (Lennox grade)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium bromide, LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>ReagentPlus, &#8805;99%</td>
		</tr>
		<tr>
			<td>Max Efficiency DH5-&#945; competent E. coli strain</td>
			<td>ThermoFisher Scientific</td>
			<td>18258012</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>MilliporeSigma</td>
			<td>322415</td>
			<td>anhydrous, 99.8%</td>
		</tr>
		<tr>
			<td>MilliQ-water</td>
			<td>EMD MilliPore</td>
			<td></td>
			<td>Milli-Q Reference Water Purification System</td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride, EDC</td>
			<td>Sigma-Aldrich</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td>average Mw ~25,000</td>
		</tr>
		<tr>
			<td>PURExpress In Vitro Protein Synthesis Kit</td>
			<td>New England BioLabs</td>
			<td>E6800S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAEX II Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>20021</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>&#160;27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-Load 2-Log DNA Ladder (0.1-10.0 kb)</td>
			<td>New England BioLabs</td>
			<td>N0469S</td>
			<td></td>
		</tr>
		<tr>
			<td>SiO&#8322; silica microspheres, 4.0 &#181;m</td>
			<td>Polysciences, Inc.</td>
			<td>24331-15</td>
			<td>10% aqueous solution</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>87724</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate, Na&#8322;CO&#8323;</td>
			<td>Sigma-Aldrich</td>
			<td>222321</td>
			<td>ACS reagent, anhydrous, &#8805;99.5%, powder</td>
		</tr>
		<tr>
			<td>Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices</td>
			<td>FisherScientific</td>
			<td>08-607-008</td>
			<td>Spectrum G235058</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase (5 U/&#181;L)</td>
			<td>ThermoFisher Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Theophylline</td>
			<td>Sigma-Aldrich</td>
			<td>T1633</td>
			<td>anhydrous, &#8805;99%, powder</td>
		</tr>
		<tr>
			<td>Tris Acetate-EDTA buffer (TAE buffer)</td>
			<td>Sigma-Aldrich</td>
			<td>T6025</td>
			<td>Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3.</td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>FisherScientific</td>
			<td>10-977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ZymoPURE II Plasmid MaxiPrep kit</td>
			<td>ZymoResearch</td>
			<td>D4202</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of multifunctional silk-based microcapsules loaded with dna plasmids encoding rna aptamers and riboswitches

Wir stellen ein Protokoll für die Herstellung von DNA-beladenen Seidenfibroin-Mikrokapseln über die Layer-by-Layer (LbL) Assemblierungsmethode auf sphärischen Opferkernen vor. Nach Adsorption einer Hauptschicht und DNA-Plasmiden wurde die Bildung robuster Mikrokapseln durch die Induktion von β-Blättern in der Seidensekundärstruktur während der akuten Dehydratisierung einer einzelnen Seidenschicht erleichtert. Daher erfolgte die Schichtung über mehrfache Wasserstoffbrückenbindungen und hydrophobe Wechselwirkungen. Nach der Adsorption von mehrschichtigen Hüllen können die Kern-Schale-Strukturen mit Goldnanopartikeln (AuNPs) und/oder Antikörpern (IgG) weiter funktionalisiert werden, um sie für die Fernerkundung und/oder den gezielten Transport zu verwenden. Die Einstellung mehrerer Schlüsselparameter während der sequentiellen Abscheidung von wichtigen Makromolekülen auf Siliziumdioxidkernen, wie z. B. das Vorhandensein eines Polymerprimers, die Konzentration von DNA und Seidenprotein sowie eine Reihe von adsorbierten Schichten, führte zu biokompatiblen, DNA-beladenen Mikrokapseln mit variabler Permeabilität und DNA-Beladung. Nach der Auflösung von Siliziumdioxidkernen zeigte das Protokoll die Bildung von hohlen und robusten Mikrokapseln mit DNA-Plasmiden, die an der Innenfläche der Kapselmembran immobilisiert waren. Durch die Schaffung einer selektiv permeablen biokompatiblen Membran zwischen den DNA-Plasmiden und der äußeren Umgebung blieb die DNA während der Langzeitlagerung erhalten und spielte eine wichtige Rolle bei der verbesserten Output-Antwort von räumlich begrenzten Plasmiden. Die Aktivität von DNA-Templates und ihre Zugänglichkeit wurden während in vitro Transkriptions- und Translationsreaktionen (zellfreie Systeme) getestet. DNA-Plasmide, die für RNA-Light-up-Aptamere und Riboschalter kodieren, wurden erfolgreich mit entsprechenden Analyten aktiviert, wie bei der Lokalisierung von fluoreszenzmarkierten RNA-Transkripten oder GFPa1-Proteinen in den Schalenmembranen sichtbar wurde.

Hier befasst sich die Studie mit der Funktionalität von DNA-Templates, die nach der Verkapselung in Seidenproteinkapseln für verschiedene Sensordesigns (zwei Arten von RNA-regulierten Transkriptions-/Translationselementen) kodieren. Mikrokapseln wurden durch Schicht-für-Schicht-Montage (LbL) der Schlüsselkomponenten hergestellt: Eine Hauptschicht, DNA-Plasmide, die für Sensordesigns kodieren, und Seidenfibroin-Biopolymer (Abbildung 2). Die schichtweise Abscheidung von Makromolekülen er...

Watch this Scientific Journal Video about Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches at JoVE.com

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Das Protokoll beschreibt die Bildung robuster und biokompatibler DNA-beladener Mikrokapseln als gemultiplexte In-vitro-Biosensoren , die in der Lage sind, mehrere Liganden zu verfolgen.

Herstellung von multifunktionalen Mikrokapseln auf Seidenbasis, die mit DNA-Plasmiden beladen sind, die für RNA, Aptamere und Riboschalter kodieren

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

preparation-multifunctional-silk-based-microcapsules-loaded-with-dna

Research

JoVE Journal

Biology

1.3K Aufrufe.  UES Inc.. Das Protokoll beschreibt die Bildung robuster und biokompatibler DNA-beladener Mikrokapseln als gemultiplexte In-vitro-Biosensoren , die in der Lage sind, mehrere Liganden zu verfolgen.

Serie: Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Hausgemachte Mutagenese von Whole Plasmide

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Forschung

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Substrat Generation für Endonukleasen der CRISPR / Cas-Systeme

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Herstellung von DNA-vernetztem Polyacrylamid, Hydrogele

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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.

Echtzeit-Bildgebung von Einzel Engineered RNA-Transkripte in lebenden Zellen Mit Ratiometrische Bimolekulare 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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Kombinierte DNA-RNA-Leuchtstoff<em&gt; In situ</em&gt; Hybridisierung (FISH) zu X-Chromosom-Inaktivierung in Differenzierte Weiblich embryonalen Stammzellen der Maus Studieren

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

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.

Reduzierte Gravitationsbereich Hardware Demonstrationen eines Prototyps Miniaturisierte Durchflusszytometer und Companion Microfluidic Mischtechnik

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Next-Generation-Sequenzierung und genomischer DNA Mapping von Retrovirale Integration Seiten

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Visualisierung von oberflächengebundenen großen DNA-Molekülen mit einem fluoreszierenden Protein-DNA-Peptid-Bindungs

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...