Figuras 1, 2, 3, 4, 6, 8 y Tablas 2, 3, 10, 15, 16 se modifican de la referencia 29. Este trabajo fue apoyado por la Fundación Nacional de Ciencia (NSF conceder-CCF 1.117.143 y NSF-CCF 1.162.141 de GS). Y.-JC fue apoyado por becas del Gobierno de Taiwán. SDR fue apoyada por el Programa Nacional Science Foundation Graduate Research Fellowship (GRFP).

<ol>
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The authors declare no competing financial interests.

Este documento describe un método para derivar puertas ndsDNA a partir de ADN plásmido de alta pureza. Por otra parte, un protocolo se presenta para caracterizar el rendimiento de puerta usando un ensayo de la cinética de fluorescencia. Los datos experimentales muestran que el sistema de plásmido derivado supera a su contraparte sintética incluso si el sistema sintético se ensambla a partir de hebras purificó usando electroforesis en gel de poliacrilamida (PAGE). Probablemente, el mejor desempeño de puertas plásmido derivado se debe principalmente a la muy alta pureza del ADN biológica. ADN sintético contiene una variedad de errores, en supresiones particulares que resultan en oligonucleótidos de longitud n-1, y dichos productos secundarios son normalmente no se elimina por completo en PAGE o cromatografía líquida de alta resolución (HPLC) procedimientos de purificación. También se observaron mejoras similares a los reportados aquí en un estudio previo de un amplificador de horquilla catalizada que utiliza ADN derivado de fuentes biológicas 21. <p clculo = &quot;jove_content&quot;&gt; Sin embargo, incluso el uso de puertas de plásmido derivado no puede eliminar por completo los errores en el desempeño de puerta, para los que hay al menos dos razones: en primer lugar el exceso de la digestión o la falta de corte de precisión puede conducir a puertas con demasiados muescas o hendiduras en las posiciones equivocadas. En cualquier caso, las puertas son más propensos a participar en reacciones no deseadas. Tales problemas pueden aliviarse mediante la optimización de la cantidad de enzima utilizada (véase la Figura 4). En segundo lugar, en estos experimentos, la mayoría de las entradas y las hebras eran auxiliares ADN sintético y de este modo contenían deleciones y mutaciones. En principio, todas las entradas de una sola hebra y hebras auxiliares también se pueden obtener a partir de ADN fagémido a través de una digestión con enzimas de nicking del genoma viral m13 pre-codificada 26. Tal vez el funcionamiento del circuito se puede mejorar adicionalmente usando ssDNA derivado del genoma bacteriano. Mientras que se encontró el uso de puertas de plásmidos derivados para mejorar el rendimiento del circuito, un análisisde los tiempos de procesamiento de coste y reveló que mientras la producción de puertas de plásmido derivado es ligeramente más barato (Tabla 15), se tarda 2-3 veces más largo el tiempo de procesamiento en comparación con el montaje y la purificación de puertas a partir de oligos sintetizados comercialmente (Tabla 16). Los costes primarios de puertas plásmido derivado son la síntesis de genes y el uso de enzimas de restricción. Por 300 pmoles de puertas (suficientes para 15 reacciones en 30 nM), el costo estimado para Únete puertas es de aproximadamente $ 170 y $ 200 para Fork puertas, la diferencia de costos se debe a la utilización de diferentes enzimas mellar. Por el contrario, la síntesis química de los hilos para los mismos costes de compuerta alrededor de $ 260 incluyendo una cuota de purificación PÁGINA. El costo de tiempo principal para puertas plásmido derivado está en el procedimiento de clonación, que, al igual que la síntesis de ADN, puede ser subcontratado a una empresa de síntesis de genes. Sin embargo, una vez ensamblados, puertas plásmido derivado tienen la ventaja de que los plásmidos de acogida fácilmente se pueden replicar unand se puede almacenar en forma de stocks de glicerol bacterianas. Esto hace posible la reutilización de las puertas muchas veces. De cara al futuro, la mejora del rendimiento de las puertas de plásmidos derivados podría permitir a una gama mucho más amplia de la dinámica de los comportamientos que han sido experimentalmente demostrado hasta ahora con ADN CRN. Por ejemplo, la reciente 47,48 trabajo teórico sugiere que los patrones espaciales auto-organizados en la escala macro se pueden realizar con el ADN CRNs a través de un mecanismo de difusión de reacción. El método que aquí se presenta proporciona una ruta viable para la construcción de los componentes moleculares subyacentes para tales materiales de ADN auto-patrones. Aunque difícil, el desarrollo de morfologías escala macro de una manera programable tendría implicaciones significativas en áreas que van desde la investigación de biomateriales para la medicina regenerativa.

La previsibilidad de Watson-Crick ha permitido a la nanotecnología de ADN dinámico para emerger como una forma programable para diseñar dispositivos moleculares con propiedades dinámicas 1,2. En particular, cadena de ADN desplazamiento - una reacción de hibridación competitiva programable - ha demostrado ser un poderoso mecanismo para la ingeniería de sistemas dinámicos de ADN. En una reacción de desplazamiento de cadena de ADN, un oligonucleótido entrante desplaza una &quot;salida&quot; hebra previamente atado de una pareja de unión complementaria. Múltiples tales reacciones pueden ser encadenados juntos en cascadas de reacción de varios pasos con un alto grado de control sobre el orden y el tiempo de reacción individual 3 pasos. ADN cascadas de desplazamiento de cadena se han utilizado para crear circuitos digitales y analógicas moleculares 4-7, 8-10 nanoestructuras conmutables, motores moleculares autónomas 11-15, y amplificadores catalíticos no covalentes 13,16-21. Por otra parte, DDispositivos NA utilizando reacciones de desplazamiento de hebra se pueden simular y diseñados para diversas aplicaciones que usan el ordenador con ayuda de software de diseño 22-24. Actualmente, el ADN sintetizado químicamente sirve como material principal para la nanotecnología de ADN. Sin embargo, los errores en el proceso de la síntesis de ADN, y los oligonucleótidos resultantes imperfectos, se cree que limitar el rendimiento de los dispositivos dinámicos de ADN al causar reacciones secundarias erróneos. Por ejemplo, las reacciones de &quot;fugas&quot; pueden resultar en la liberación de un oligonucleótido de salida incluso en la ausencia de un disparador de reacción. Estos efectos son más evidentes en las cascadas de reacción autocatalíticos donde incluso una cantidad mínima de fuga inicial con el tiempo como resultado la activación completa del 19,20 cascada. Por el contrario, las reacciones a menudo fallan en alcanzar el nivel esperado de activación debido a que algunos componentes no activan incluso en la presencia de la entrada prevista 7,25. Para hacer que el rendimiento de ADN a base denanodispositivos comparables a sus homólogos basados ​​en proteínas biológicas, tales modos de error deben reducirse dramáticamente. Plásmidos bacterianos u otro ADN biológica podrían servir como una fuente relativamente barata de ADN de alta pureza para aplicaciones de la nanotecnología. Grandes cantidades de ADN se pueden generar por la replicación en bacterias y las capacidades de corrección de pruebas intrínsecas de los sistemas vivos asegurar la pureza del ADN resultante. De hecho, varios estudios recientes han reconocido la utilidad potencial de ADN biológico para aplicaciones de la nanotecnología 21,26-28. Sin embargo, la naturaleza completamente de doble hebra de ADN del plásmido hasta ahora ha prohibido su uso como material para la fabricación de dispositivos dinámicos de ADN, que normalmente consisten en múltiples oligonucleótidos y contienen ambos dominios de doble cadena y de cadena sencilla. En un trabajo reciente 29 de este tema fue abordado y una nueva arquitectura de puerta de ADN que se compone principalmente de ADN de doble cadena mellado (ndsDNA) fue introducirre. Es importante destacar que los sistemas de puertas ndsDNA pueden ser diseñados que dan cuenta de la dinámica especificados por cualquier red reacción química formales (CRN) 29. puertas ndsDNA tanto, se podrían utilizar, en principio, para crear sistemas dinámicos que presentan oscilaciones y caos, biestabilidad y la memoria, la lógica de Boole o comportamientos algorítmicos 30-38. Por ejemplo, Ref. 29 demostraron una CRN de tres reacción que proporciona una implementación molecular de un protocolo de &quot;consenso&quot;, un tipo de algoritmo de computación distribuida 29,39,40. Este trabajo demuestra por primera vez un nuevo uso para el formalismo CRN como un &quot;lenguaje de programación&quot; para sintetizar rápidamente los sistemas moleculares funcionales (Figura 1). Aquí, se proporciona un protocolo detallado para derivar puertas ndsDNA de ADN plásmido. Primero es una revisión del proceso de diseño de la secuencia. Luego sigue una explicación de cómo oligonucleótidos sintéticos que contienenlas secuencias de compuerta se clonaron en plásmidos y la secuencia verificados y amplificados a través de cultivo bacteriano. A continuación, se muestra cómo ndsDNA puertas se pueden derivar de los plásmidos mediante procesamiento enzimático (véase la Figura 2). Finalmente, un método para probar el comportamiento puerta utilizando ensayos de cinética de fluorescencia se describe. Mecanismo de reacción Como ejemplo, el protocolo se centra en el catalítica reacción química A + B-&gt; B + C. La especie A, B, y C (&quot;señales&quot;, Figura 1B) todos corresponden a una molécula de ADN de una sola hebra diferente. Las secuencias de estas moléculas son completamente independientes y las hebras no reaccionan entre sí directamente. Las secuencias de todas las señales tienen dos dominios funcionales diferentes, es decir, subsecuencias que actúan juntos en reacciones de desplazamiento de hebra: 1) un dominio de punto de apoyo corto (etiquetas ta, tb, tc) que se utiliza para la iniciación de desplazamiento de cadena reaction, y 2) un dominio de largo (etiquetas a, b, c) que determina la identidad de la señal. Las interacciones entre los hilos de señal están mediadas por ADN (ndsDNA) complejos de puerta de doble cadena con muescas (llamados Únete AB y Tenedor aC) y las especies de cadena sencilla auxiliares (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; y &lt;i tc &gt;). La reacción A + B- formales&gt; B + C se ejecuta a través de una serie de etapas de reacción de desplazamiento de cadena, donde cada paso de reacción expone un punto de apoyo para una reacción posterior (Figura 1B). En este ejemplo, las señales A y B son inicialmente libre en solución, mientras que la señal C está unido a la puerta de tenedor. Al final de la reacción B y C están en solución. De manera más general, las señales que están obligados a una puerta están inactivas mientras que las señales que están libres en solución son activos, es decir, que pueden participar en una reacción de desplazamiento de cadena comouna entrada. El curso temporal de la reacción se sigue utilizando una estrategia de indicador fluorescente (Figura 1C). En un trabajo anterior 29, se demostró que este mecanismo de reacción no sólo da cuenta de la estequiometría correcta, sino también la cinética de la reacción objetivo.

<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.

Plásmido derivado de ADN Strand Displacement Puertas de Redes de reacción de Ejecución Química

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 vistas.  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. 

Video: 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.

El análisis de ADN de doble cadena Break (OSD) de reparación en células de mamífero

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 ...

Investigación

Biología

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.

Cómo Cultura, Registro y estimular las redes neuronales en matrices de micro-electrodos (MEA)

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.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

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.

Iterativo de optimización de dúplex de ADN para la cristalización de SEQA-ADN complejos

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.

La identificación de bacterias metabólicamente activas en el intestino de la Generalista<em&gt; Spodoptera littoralis</em&gt; Vía ADN isótopos estables Uso<sup&gt; 13</sup&gt; C-glucosa

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.

Los protocolos para la implementación de un<em&gt; Escherichia coli</emSistema&gt; Basado TX-TL-Cell de Libre Expresión de la biología sintética

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.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

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.

Reducción de gravedad Demostraciones entorno de hardware de un prototipo miniaturizado citómetro de flujo y Compañero Microfluidos Tecnología de mezcla

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.

Reacción seleccionada Monitoreo espectrometría de masas para Absolute Proteína cuantificación

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.

En Vivo alcalina Comet Assay y Comet Assay alcalina modificada con enzimas para la medición de roturas en el ADN Strand y daño oxidativo del ADN en el hígado de rata

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