Figuren 1, 2, 3, 4, 6, 8 und den Tabellen 2, 3, 10, 15, 16 sind aus Lit. 29 modifiziert. Diese Arbeit wurde von der National Science Foundation (NSF gewähren CCF-1.117.143 und NSF-CCF 1.162.141 auf GS). Y.-JC wurde vom taiwanesischen Regierung Stipendien unterstützt. SDR wurde von der National Science Foundation Graduate Research Fellowship Program (GRFP) unterstützt.

<ol>
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The authors declare no competing financial interests.

Dieser Artikel beschreibt ein Verfahren zum Ableiten ndsDNA Gates aus hochreiner Plasmid-DNA. Darüber hinaus wird ein Protokoll zur Charakterisierung Gate Leistung durch einen Fluoreszenz-Assay-Kinetik dargestellt. Experimentelle Daten zeigen, dass das Plasmid stammSystem trifft sein synthetisches Gegenstück, selbst wenn das synthetische System ist aus Strängen mit Polyacrylamid-Gelelektrophorese (PAGE) gereinigt, montiert. Wahrscheinlich ist die verbesserte Leistung von Plasmid stammende Gates vor allem auf die hohe Reinheit der biologischen DNA. Synthetische DNA enthält eine Vielzahl von Fehlern, insbesondere Deletionen, die Oligonukleotide mit einer Länge von n-1, und solche Nebenprodukte entstehen, werden in der Regel nicht vollständig PAGE oder Hochleistungs-Flüssigkeits-Chromatographie (HPLC) Reinigungsverfahren entfernt. Ähnliche Verbesserungen bei den hier berichteten wurden in einer früheren Studie von einer katalysierten Haarnadel-Verstärker, der aus biologischen Quellen 21 abgeleitete DNA verwendet beobachtet. <p class = &quot;jove_content&quot;&gt; Aber auch die Verwendung von Plasmid-abgeleitete Gatter können nicht vollständig beseitigen Fehler in der Gate Leistung, für die es mindestens zwei Gründe: Erstens über Verdauung oder Mangel an Schnittgenauigkeit können die Gates mit zu vielen führen Einschnitte oder Kerben in den falschen Positionen. In jedem Fall sind Gates eher in unerwünschter Reaktionen teilzunehmen. Solche Probleme können durch eine Optimierung der Menge des verwendeten Enzyms (siehe Abbildung 4) gelockert werden. Zweitens, in diesen Experimenten am Ein- und Hilfsstränge wurden synthetische DNA und enthielt Deletionen und Mutationen bei. Prinzipiell sind alle Einzelstrang Antriebs- und Hilfs Stränge könnte auch aus Phagemid-DNA durch eine Nicking-Enzym-Verdauung des vorcodierten m13 Virusgenom 26 erhalten werden. Vielleicht die Leistungsfähigkeit der Schaltung kann ferner unter Verwendung von ssDNA aus dem Bakteriengenom abgeleitet verbessert werden. Während die Verwendung von Plasmid-abgeleitete Gattern wurde gefunden, daß die Schaltungsleistung zu verbessern, eine Analyseder Kosten und Bearbeitungszeiten ergeben, dass, während die Herstellung von Plasmid stammende Gates etwas günstiger ist (Tabelle 15), dauert es 2-3 mal längere Verarbeitungszeit im Vergleich zur Montage und Reinigung von Gattern aus kommerziell synthetisiert Oligos (Tabelle 16). Die primären Kosten von Plasmid stammende Gates Gensynthese und die Verwendung von Restriktionsenzymen. 300 pmol von Gattern (ausreichend für 15 Reaktionen bei 30 nM), die geschätzten Kosten für die Join-Gatter ist ungefähr $ 170 und $ 200 für Gabel Tore, die Kostendifferenz wobei aufgrund der Verwendung von unterschiedlichen Nicking-Enzyme. Im Gegensatz dazu ist die chemische Synthese der Stränge für die gleiche Gate kostet rund 260 $ einschließlich einer PAGE Reinigungsgebühr. Die primären Zeitkosten für Plasmid stamm Toren ist in der Klonierung, die, genau wie die DNA-Synthese, kann zu einer Gensynthese Unternehmen ausgelagert werden. Haben jedoch nach der Montage Plasmid stamm Gatter den Vorteil, dass der Host-Plasmide können leicht zu einem replizierendennd in Form von bakteriellen Glycerinstamm gespeichert werden. Dadurch ist es möglich, um die Gates mehrfach wieder verwenden. Wir freuen uns, könnte die verbesserte Leistung von Plasmid stamm Toren einen wesentlich größeren Dynamikbereich als Verhaltensweisen sind experimentell bisher mit DNA CRNs gezeigt zu ermöglichen. Zum Beispiel schlug den letzten theoretischen Arbeit 47,48, dass selbstorganisierte räumliche Muster auf der Makroskala kann mit DNA CRNs durch eine Reaktionsdiffusionsmechanismus realisiert werden. Die hier vorgestellte Methode bietet eine praktikable Weg zum Aufbau der zugrunde liegenden molekularen Komponenten für solche Selbst Strukturieren DNA Materialien. Obwohl anspruchsvoll, die Entwicklung Makromaßstab Morphologien in einer programmierbaren Weise würde erhebliche Auswirkungen in Bereichen wie Biomaterialforschung an der regenerativen Medizin haben.

Die Vorhersehbarkeit der Watson-Crick-Basenpaarung konnten dynamischen DNA-Nanotechnologie als eine programmierbare Weg zu molekularen Geräte mit dynamischen Eigenschaften 1,2 entwerfen entstehen. Insbesondere DNA Strangverdrängung - eine programmierbare, wettbewerbsfähige Hybridisierungsreaktion - hat sich als leistungsfähigen Mechanismus für das Engineering dynamischer DNA Systeme sein. In einem DNA-Strang Verdrängungsreaktion, verdrängt ein eingehender Oligonukleotid eine zuvor gebunden &quot;output&quot; Strang aus einem komplementären Bindungspartner. Mehrere solche Reaktionen können zusammen in mehrstufigen Reaktorkaskaden mit einem hohen Grad an Kontrolle über die Reihenfolge und das Timing der einzelnen Reaktionsschritte 3 verkettet werden. DNA-Strangverdrängungs Kaskaden verwendet worden, um digitale und analoge Schaltungen Molekular 4-7, umschaltbar Nanostrukturen 8-10 autonome molekulare Motoren 11-15, und nicht-kovalente katalytischer Verstärker 13,16-21 erstellen. Außerdem DNA Geräte mit Strangverdrängungsreaktionen können simuliert und für vielfältige Anwendungen unter Verwendung von computergestützten Design-Software 22-24 ausgelegt sein. Derzeit ist chemisch synthetisierte DNA als Hauptmaterial für die DNA-Nanotechnologie. Jedoch Fehler in der DNA-Syntheseverfahren und die so erhaltenen unvollkommen Oligonukleotiden wird angenommen, dass die Leistung von dynamischen DNA von Geräten durch fehlerhafte Nebenreaktionen zu begrenzen. Zum Beispiel &quot;Leck&quot; Reaktionen können in der Freisetzung eines Ausgangs Oligonucleotid selbst in der Abwesenheit eines Reaktionstrigger führen. Solche Effekte werden in autokatalytischen Reaktionskaskaden, wo auch nur eine minimale Menge von anfänglichen Leck wird schließlich in die vollständige Aktivierung des Kaskaden 19,20 führen offensichtlichsten. Umgekehrt Reaktionen oft nicht zu dem erwarteten Ausmaß der Aktivierung zu erreichen, da einige Komponenten nicht einmal in Anwesenheit von die beabsichtigte Eingabe 7,25 auszulösen. Um die Leistung zu machen DNA basierendenNanovorrichtungen vergleichbar zu biologischen Proteinbasis benötigen solche Fehlerarten, drastisch reduziert werden. Bakterielle Plasmide oder andere biologische DNA konnte als relativ billige Quelle für hochreine DNA für Nanotechnologie-Anwendungen dienen. Große Mengen von DNA können durch Replikation in Bakterien erzeugt werden, und die Grenzkorrekturlesen Fähigkeiten lebender Systeme sorgen für die Reinheit des erhaltenen DNA. In der Tat haben einige neue Papiere den potenziellen Nutzen der biologischen DNA für Nanotechnologie-Anwendungen 21,26-28 anerkannt. Jedoch die vollständig doppelsträngige Art der Plasmid-DNA hat bisher die Verwendung als Material zur Herstellung von dynamischen DNA-Geräte, die typischerweise aus mehreren Oligonukleotiden und enthalten sowohl doppelsträngige und einzelsträngige Bereiche gestattet. In einem kürzlich erschienenen Papier 29 wurde diese Frage angesprochen und eine neue DNA-Gate-Architektur, die in erster Linie aus geklaut doppelsträngige DNA (ndsDNA) aus war einzuführend. Wichtig ist, dass Systeme der ndsDNA Tore ausgelegt sein, dass die durch jede formale chemische Reaktion Netzwerk (CRN) 29 angegeben Dynamik zu realisieren. ndsDNA Gates könnte also verwendet werden, grundsätzlich auf dynamische Systeme, die Schwingungen und Chaos, Bistabilität und Gedächtnis, Boolesche Logik oder algorithmische Verhaltensweisen 30-38 aufweisen erstellen. Zum Beispiel 29 Ref. Gezeigt, eine dreiReaktions CRN, die ein Molekular Implementierung eines &quot;Konsensus&quot; Protokoll an, das der verteilten Rechenalgorithmus 29,39,40 vorgesehen. Diese Arbeit erstmals gezeigt werden eine neue Verwendung für die CRN-Formalismus als eine &quot;Programmiersprache&quot; zur schnellen Synthese von funktionalen molekularen Systemen (Abbildung 1A). Hier ist ein detailliertes Protokoll zum Ableiten ndsDNA Gates von Plasmid-DNA ist. Zunächst ist eine Überprüfung der Sequenz Design-Prozess. Es folgt eine Erläuterung, wie synthetische Oligonukleotide enthaltendie Gate-Sequenzen in Plasmide kloniert und die Sequenz verifiziert und durch Bakterienkultur amplifiziert. Als nächstes wird gezeigt, wie ndsDNA Tore können aus den Plasmiden durch enzymatische Verarbeitung abgeleitet werden (siehe Abbildung 2). Schließlich wird ein Verfahren zum Testen von Gate Verhaltens mit Fluoreszenzkinetiken Assays beschrieben. Reaktionsmechanismus Als ein Beispiel setzt das Protokoll auf die katalytische chemische Reaktion A + B-&gt; B + C. Die Arten A, B und C (&quot;Signale&quot;, 1B) entsprechen alle einem anderen Einzelstrang-DNA-Moleküls. Die Sequenzen dieser Moleküle sind völlig unabhängig und die Stränge nicht direkt miteinander reagieren. Die Sequenzen aller Signale weisen zwei verschiedene funktionelle Domänen, das heißt, Teilsequenzen, die in Strangverdrängungsreaktionen zusammenwirken: 1) eine kurze toehold Domäne (labels ta, tb, tc), die für die Initiation der Strangverdrängung r verwendet wirdeAction, und 2) eine lange Domain (Etiketten a, b, c), die das Signal Identität bestimmt. Wechselwirkungen zwischen Signalstränge durch geklaut doppelsträngige DNA (ndsDNA) Gate-Komplexe vermittelte (genannt anmelden AB und Gabel BC) und Hilfseinzelstrang-Arten (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; und &lt;i tc &gt;). Die formale Reaktions A + B-&gt; B + C wird durch eine Reihe von Strangverdrängungsreaktionsschritten, wobei jeder Reaktionsschritt setzt einen Ansatzpunkt für eine anschließende Reaktion (1B) ausgeführt. In diesem Beispiel werden Signale A und B sind anfänglich frei in Lösung, während das Signal C dem Gabel Gate gebunden. Am Ende der Reaktion B und C sind in Lösung. Allgemeiner Signale, die an ein Gate gebunden sind inaktiv, während Signale, die frei in Lösung aktiv sind, das heißt, sie können in einer Strangverdrängungsreaktion als teilnehmenein Eingang. Der zeitliche Verlauf der Reaktion wird unter Verwendung eines fluoreszierenden Reporterstrategie (Abbildung 1C), gefolgt. In früheren Arbeiten 29 wurde gezeigt, dass diese Reaktion Mechanismus realisiert sowohl die korrekte Stöchiometrie als auch die Kinetik der Zielreaktion.

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

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

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

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

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

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

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

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

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

Research

JoVE Journal

Biology

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

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

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

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

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

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

Die Analyse der DNA-Doppelstrang Break (DSB) Reparatur in Säugerzellen

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

Forschung

Biologie

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

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

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

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

Wie Kultur, Record und stimulieren Neuronale Netze auf Micro-Elektroden Arrays (MEAs)

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

Bioengineering Human Microvascular Networks in Immunodeficient Mice

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

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

Bioengineering humane mikrovaskuläre Networks in immundefizienten Mäusen

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

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

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

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

Iterative Optimierung der DNA-Duplexes für Kristallisation von SeqA-DNA-Komplexen

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

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

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

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

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

Bezeichnung des metabolisch aktiven Bakterien im Darm der Generalist<em&gt; Spodoptera littoralis</em&gt; Über DNA Stable Isotope Probing Mit<sup&gt; 13</sup&gt; C-Glucose

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

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

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

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

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

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 ...

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 ...

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 ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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

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

Selected Reaction Monitoring-Massenspektrometrie für Absolute Proteinquantifizierung

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

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

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

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

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

In - vivo - Alkaline Comet Assay und enzymmodifizierten Alkaline Comet Assay zur Messung von DNA - Strangbrüchen und oxidative DNA - Schäden in der Rattenleber

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