As Figuras 1, 2, 3, 4, 6, 8 e quadros 2, 3, 10, 15, 16 são modificados a partir de 29 Ref. Este trabalho foi financiado pela National Science Foundation (NSF conceder-CCF 1117143 e NSF-CCF 1.162.141 para GS). Y.-JC foi apoiado por bolsas de estudo governamentais de Taiwan. SDR foi apoiada pela Fundação Nacional de Ciência Programa de Pós-Graduação Research Fellowship (GRFP).

<ol>
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The authors declare no competing financial interests.

Este artigo descreve um método para derivar portões ndsDNA a partir de DNA de plasmídeo muito puro. Além disso, um protocolo é apresentada para caracterizar o desempenho portão utilizando um ensaio de cinética de fluorescência. Os dados experimentais mostram que o sistema de plasmídeo derivado de supera sua contraparte sintética, mesmo se o sistema sintético é montado a partir de fios purificado utilizando electroforese em gel de poliacrilamida (PAGE). Provavelmente, o melhor desempenho de portas derivado do plasmídeo é principalmente devido à muito alta pureza do DNA biológico. ADN sintético contém uma variedade de erros, em particular, deleções que resultam em oligonucleótidos de tais produtos secundários comprimento n-1, e são tipicamente não completamente removido em PAGE ou cromatograf ia líquida de alta eficiência (HPLC) procedimentos de purificação. Também foram observadas melhorias semelhantes aos aqui relatados em um estudo prévio de um amplificador hairpin catalisada que usou DNA derivado de fontes biológicas 21. <p class = &quot;jove_content&quot;&gt; No entanto, mesmo o uso de portas derivado do plasmídeo não pode eliminar completamente os erros no desempenho portão, para as quais existem pelo menos duas razões: em primeiro lugar sobre-digestão ou falta de precisão de corte pode levar a portões com demasiados nicks ou cortes nas posições erradas. Em qualquer caso, as portas são mais susceptíveis de participar em reacções indesejadas. Tais problemas podem ser facilitado através da optimização da quantidade de enzima utilizada (ver Figura 4). Em segundo lugar, nestas experiências, a maioria das entradas e cordões eram auxiliares de ADN sintético e continha assim as supressões e mutações. Em princípio, todas as entradas de cadeia simples e fios auxiliares também poderia ser obtido a partir de ADN de fagemideo por meio de uma digestão com enzimas de nicking do genoma viral M13 pré-codificada 26. Talvez o desempenho do circuito pode ser melhorado utilizando ADNcs derivados do genoma bacteriano. Enquanto o uso de portões derivado de plasmídeo foi encontrada para melhorar o desempenho do circuito, uma análisedos tempos de processamento e custos revelou que, enquanto a produção de portas derivado de plasmídeo é ligeiramente mais barato (Tabela 15), leva-se 2-3 vezes mais em comparação com o tempo de processamento e a purificação da montagem de portas de oligos sintetizados comercialmente (Tabela 16). Os custos primários de portões são derivados do plasmídeo síntese de genes e a utilização de enzimas de restrição. Para 300 pmol de portas (suficiente para 15 reacções em 30 nM), o custo estimado para Junte-se portas é de aproximadamente US $ 170 e US $ 200 para Fork portões, a diferença de custo ser devido ao uso de diferentes enzimas nicking. Em contraste, a síntese química dos fios para os mesmos custos de porta em torno $ 260, incluindo uma taxa de purificação PAGE. O custo do tempo primário para portas derivado do plasmídeo é no procedimento de clonagem, que, assim como a síntese de DNA, pode ser terceirizado para uma empresa de síntese de genes. No entanto, uma vez montado, portas derivado do plasmídeo tem a vantagem de que os plasmídeos hospedeiros podem ser facilmente replicadas de umaND pode ser armazenado na forma de estoques de glicerol bacterianas. Isto faz com que seja possível voltar a utilizar os portões muitas vezes. Olhando para a frente, o melhor desempenho de portas derivado do plasmídeo poderia permitir uma gama muito maior de dinâmica de comportamentos que têm sido experimentalmente demonstrado até agora com DNA CRNs. Por exemplo, recente trabalho teórico 47,48 sugeriu que padrões espaciais auto-organizadas na macro-escala podem ser realizados com DNA CRNs através de um mecanismo de difusão reação. O método aqui apresentado fornece um caminho viável para a construção de componentes moleculares subjacentes a tais materiais de DNA auto-padronização. Apesar de desafiador, desenvolvendo morfologias macro-escala de uma forma programável teria implicações significativas em áreas que vão desde pesquisas em biomateriais para a medicina regenerativa.

A previsibilidade de bases de Watson-Crick tem permitido nanotecnologia de DNA dinâmico a emergir como uma forma programável para projetar dispositivos moleculares com propriedades dinâmicas 1,2. Em particular, o deslocamento cadeia de DNA -, uma reação de hibridização competitiva programável - provou ser um poderoso mecanismo de engenharia de sistemas dinâmicos de DNA. Em uma reação de deslocamento de cadeia de DNA, um oligonucleï¿½ido de entrada desloca um &quot;output&quot; vertente anteriormente vinculado a partir de um parceiro de ligação complementar. Várias tais reações podem ser encadeados em cascatas de reação de várias etapas com um alto grau de controle sobre a ordem eo tempo de reação individual etapas 3. DNA cascatas de deslocamento de cadeia foram usadas para criar circuitos digitais e analógicos moleculares 4-7, nanoestruturas comutáveis ​​8-10, motores moleculares autónomas 11-15, e amplificadores catalíticos não covalentes 13,16-21. Além disso, DNA dispositivos que utilizam reações de deslocamento de cadeia pode ser simulado e projetado para diversas aplicações utilizando software 22-24 desenho assistido por computador. Atualmente, o DNA sintetizado quimicamente serve como o principal material para a nanotecnologia de DNA. No entanto, os erros no processo de síntese de DNA, e os oligonucleótidos imperfeitos resultantes, são acreditados para limitar o desempenho de dispositivos de ADN dinâmicas, fazendo com que as reacções laterais erradas. Por exemplo, as reacções de &quot;vazamento&quot; pode resultar na libertação de um oligonucleótido de saída, mesmo na ausência de uma reacção de disparo. Tais efeitos são mais óbvias nas cascatas de reacção autocatalítica onde mesmo uma quantidade mínima de vazamento inicial irá eventualmente resultar na activação completa da cascata 19,20. Por outro lado, as reacções muitas vezes não conseguem alcançar o nível esperado de activação porque alguns componentes não provocam, mesmo na presença de 7,25 a entrada pretendida. Para fazer com que o desempenho de DNA com base emnanodispositivos comparáveis ​​com os seus homólogos à base de proteínas biológicas, tais modos de erro precisa ser reduzido drasticamente. Plasmídeos bacterianos ou outro ADN biológica pode servir como uma fonte de ADN altamente puro relativamente barato para aplicações de nanotecnologia. Grandes quantidades de ADN pode ser gerado por replicação em bactérias e as capacidades inerentes de revisão dos sistemas vivos garantir a pureza do ADN resultante. De fato, vários trabalhos recentes têm reconhecido a utilidade potencial de DNA biológico para aplicações de nanotecnologia 21,26-28. No entanto, a natureza totalmente de cadeia dupla de DNA de plasmídeo foi medida proibida a sua utilização como um material para a fabricação de dispositivos de ADN dinâmicas, que consistem tipicamente de vários oligonucleótidos e contêm ambos os domínios de cadeia dupla e de cadeia simples. Em um recente artigo 29 esta questão foi abordada e uma nova arquitetura portão DNA que consiste principalmente de DNA de cadeia dupla entalhado (ndsDNA) foi introduzird. É importante ressaltar que os sistemas de portões ndsDNA pode ser concebido que perceber a dinâmica especificados por qualquer rede de reação química formais (CRN) 29. portões ndsDNA poderia, assim, ser utilizados, em princípio, para criar sistemas dinâmicos que apresentam oscilações e caos, biestabilidade e memória, lógica booleana ou comportamentos algorítmicos 30-38. Por exemplo, Ref. 29 demonstraram uma CRN três reacção que fornecida uma implementação molecular de um protocolo de &quot;consenso&quot;, um tipo de algoritmo de computação distribuída 29,39,40. Este trabalho demonstrou pela primeira vez uma nova utilização para o formalismo CRN como uma &quot;linguagem de programação&quot; para sintetizar rapidamente sistemas moleculares funcionais (Figura 1A). Aqui, um protocolo detalhado para derivar portões ndsDNA de ADN de plasmídeo é fornecida. Primeiro é uma revisão do processo de concepção sequência. Depois segue-se uma explicação de como oligonucleótidos sintéticos que contêmas sequências de porta são clonados em plasmídeos e sequência verificada e amplificado através de cultura bacteriana. Em seguida, é mostrado como ndsDNA portas podem ser derivados a partir dos plasmídeos por tratamento enzimático (ver Figura 2). Por fim, um método para testar o comportamento portão usando a cinética de fluorescência os ensaios é descrita. Mecanismo de reacção Como um exemplo, o protocolo centra-se na reacção química catalítica de A + B&gt; B + C. As espécies de A, B, e C (&quot;sinais&quot;, Figura 1B) correspondem todas a uma molécula diferente de cadeia simples de ADN. As sequências destas moléculas são completamente independentes e os fios não reagem um com o outro directamente. As sequências de todos os sinais têm dois domínios funcionais diferentes, ou seja, subsequências que agem em conjunto em reacções de deslocamento de cadeia: 1) um ponto de apoio de domínio curto (etiquetas TA, TB, TC) que é usado para a iniciação da cadeia deslocamento reaction, e 2) um domínio longo (etiquetas a, b, c) que determina a identidade do sinal. Interações entre fios de sinal são mediados por ADN (ndsDNA) Complexos de porta de cadeia dupla com entalhe (chamados Adira AB e Fork BC) e as espécies de cadeia simples auxiliares (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; e &lt;i tc &gt;). A reacção de formal de A + B&gt; B + C é executada através de uma série de passos de reacção de deslocamento de cadeia, em que cada passo reaccional expõe um ponto de apoio para uma reacção subsequente (Figura 1B). Neste exemplo, os sinais A e B são inicialmente livre em solução enquanto o sinal de C está ligado ao portão forquilha. No final da reacção, B e C estão em solução. De modo mais geral, os sinais que estão vinculados a um portão estão inativos, enquanto os sinais que são livres em solução são ativos, isto é, eles podem participar de uma reação de deslocamento de cadeia comouma entrada. O curso do tempo da reacção é seguido usando uma estratégia repórter fluorescente (Figura 1C). Em trabalho anterior 29, demonstrou-se que este mecanismo de reacção, não só realiza a estequiometria correcta, mas também a cinética da reacção alvo.

<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.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

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

14.1K visualizações.  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. 

Vídeo: 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.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

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 ...

Biologia

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.

Como gravar, Cultura e estimular redes neuronais em Micro-Arrays eletrodo (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.

Bioengenharia Humanos Networks microvascular em camundongos imunodeficientes

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 Otimização de Duplexos de DNA para Cristalização de SEQA-Complexos de DNA

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.

Identificação de bactérias metabolicamente ativas no intestino do Generalista<em&gt; Spodoptera littoralis</em&gt; Via DNA Isótopos Estáveis ​​Sondagem Usando<sup&gt; 13</sup&gt; C-Glicose

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.

Protocolos para implementação de uma<em&gt; Escherichia coli</em&gt; Com base TX-TL Expression System-Free Cell para Biologia 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.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica 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 ...

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.

De gravidade reduzida Manifestações ambiente de hardware de uma tecnologia de mistura de fluxo Cytometer e Companion microfluídicos Prototype Miniaturized

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.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

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 Ensaio Cometa e Enzyme-modificado Alkaline Comet Ensaio para medição Breaks DNA Strand e dano oxidativo ao DNA em Rat Liver

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