Os autores gostariam de agradecer a Jen Sheen do Hospital Geral de Massachusetts para a generosa doação de mudas de Nicotiana benthamiana. Jen Bush nos ajudou muito no conselho em cultivo de plantas ea criação de uma área de câmara de crescimento. Tom Ferrante, do Instituto Wyss ofereceu ajuda fundamental com microscopia confocal. Os autores gostaria especialmente de agradecer Don Ingber do Hospital Infantil de Boston e do Instituto Wyss para a generosa doação de um Gene Gun e suprimentos associados. O financiamento para este projeto foi fornecido por meio de um acordo de cooperação com o Departamento de Energia Projetos de Pesquisa Avançada Agência (ARPA-E Award # DE-000079) para PAS, JCW, e MdM, e através Chimerion Biotechnology, Inc. para MJV.

1. Projeto de Seqüências Alternativamente-emendados para vários Organelle Segmentação <ol><li> Determinar os locais de expressão de proteína final. Para este trabalho, o interesse é na segmentação do cloroplasto, peroxissomo e citosol. </li><li> Use a literatura para identificar seqüências de proteínas e de DNA conhecidos como alvo proteínas para organelas de interesse. Neste caso, uma sequência de direccionamento a partir de cloroplastos de Arabidopsis thaliana L-isoaspartate metiltransferase 6,15, e uma sequência de direccionamento de peroxissoma de Arabidopsis thaliana, como a transtirretina-S-alantoína sintase 16,17 foram determinados. </li><li> Identificar um regime-splicing alternativo que deve direcionar as proteínas de interesse para as organelas corretas. Para este trabalho, splice sites, observada num sistema de Arabidopsis naturais 6. A seqüência de mRNA foi redesenhado deixando os locais de splicing naturais, mas as sequências foram colocadas de codificação-cloroplastos ou peroxisome-targeting tags dentro dos íntrons alternativamente-emendados (Figura 1). </li></ol> 2. Assembléia Gibson de Peças de DNA Veja também a Figura 2. O método de montagem Gibson depende da ação de várias enzimas em uma mistura pré-fabricados a 1) digerir parcialmente as extremidades do DNA de cadeia dupla de fazer cadeias simples e 2) anneal pares de bases de cortesia da vizinha partes de DNA. Isto permite a clonagem de fragmentos de DNA, sem as limitações de locais de restrição. <ol><li> Escolher uma ordem para os elementos na construção de plasmídeo de ADN. Como exemplo TriTag-1 tem elementos de: a) um vector de plasmídeo (poro, contendo uma construção de GFP conhecido para trabalhar em plantas, um marcador de resistência a canamicina, e origens de replicação para E. coli e Agrobacterium tumefaciens hospedeiros), b) um sequência de promotor, (P ENTCUP2, expressa constitutivamente em plantas), C) uma sequência de direccionamento do cloroplasto (CTS), c) uma sequência de peroxissoma (PTS), e d alvo) de uma série de locais de união, tal como descrito anteriormente 6. </li><li> Projetar o base-para-base de construção com em software de projeto silico. Macaco é um pacote gratuito de código aberto útil para este trabalho. Nota: TriTag-1, tem a ordem: plasmídeo vetor, promotor, ATG, local dador, seqüência alvo cloroplasto, área doadora emenda, local neutro, local aceitante emenda, seqüência alvo peroxisome, local aceitante emenda, GFP como contido no plasmídeo vetor . <ol><li> Projetar o in silico construção de DNA de tal forma que há uma sobreposição de 50 pb entre cada peça ao encomendar primers para PCR. É possível usar a sobreposição de 50 pb, como iniciadores: 25 pb cada direcção. <ol><li> Certifique-se de manter o controle da origem de cada uma das peças de seqüência. É como: um plasmídeo a ser cultivadas e miniprepped; uma sequência linear que podem ser utilizados como um molde de PCR; ou uma sequência que terá de sersintetizado comercialmente? Várias empresas oferecem serviços de síntese de DNA de baixo custo para os fragmentos &lt;500 pb. Neste caso, o próprio TriTag é de 437 pb, pelo que foi vantajoso por ordem comercialmente. </li><li> Os melhores resultados virão se todos os fragmentos são amplificados por PCR e purificou-se a partir deles o DNA molde. O marcador de resistência é um bom local para dividir esta grande de ADN em dois modelos menores, que são mais facilmente amplificado por PCR. </li><li> Enzima de restrição Dpnl restrição corta DNA metilado. Se o molde de PCR era uma miniprep de E. coli, o tratamento Dpnl irá remover o ADN molde contaminante. </li></ol></li></ol></li><li> Purificar todas as seqüências de DNA separadamente e eluição em água, TE ou tampão EB. <ol><li> Pore ​​vetor parte 1, ~ 3000 pb (setas verdes). Amplificado por PCR e Dpnl tratada. </li><li> Pore ​​vetor parte 2, ~ 3000 pb (setas pretas). Amplificado por PCR e Dpnl tratada. </li><li> TriTag inserção, 437 bp (setas azuis). Ordenada comercialmente. Resuspend em DDH 2 O. </li><li> P ENTCUP promotor, ~ 750 bp (setas laranja). Amplificado por PCR e Dpnl tratada. </li></ol></li><li> Medem-se as concentrações de cada uma das partes de ADN. Apontar para 150 ng / mL das peças vetoriais. </li><li> Tome uma alíquota do mix montagem Gibson fora do freezer e descongelar em gelo. Isto está comercialmente disponível, mas também simples de fazer no laboratório 12-14. <ol><li> Há duas etapas para esta receita: um master mix 5x viscoso e 15 ul alíquotas 1.33x reação porte. A mistura principal 5x contém: 3 ml de 1 M de Tris-HCl pH 7,5, 300 ul de 1 M de MgCl2, 600 ul de 10 mM de cada dNTP, 300 ul de 1 M DTT, 1,5 g de PEG-8000, 20 mg de NAD, e ddH 2 O 6 ml. Prepare 320 mL alíquotas (18) e congelar todos, mas um a -20 ° C. A solução irá ser bastante viscoso. Rotular essas &quot;tampão isotérmica 5X&quot; </li><li> Para o restante (320 mL), adicionar 1,2 mL T5 exonuclease, 20 ul Phusion alta-FIDElity DNA polimerase, 160 uL de Taq ADN ligase e 700 ul ddH 2 O. Preparar 15 mL alíquotas (~ 80) em gelo em tubos de PCR e armazenar a -20 ° C. </li></ol></li><li> Determinar os volumes de quantidades equimolares de cada um dos fragmentos. Existem algumas calculadoras on-line, incluindo Gibthon.org. Deve haver um total de 5 mL de todas as partes de ADN. Adicione-os a 15 mL Gibson misturar alíquota. Se isto requer volumes muito pequenos a pipeta, diluir os fragmentos de DNA e / ou de usar mais do que uma alíquota. <ol><li> Não se esqueça de preparar um controle do DNA vetor sozinho (por exemplo, a parte do DNA que contém o marcador de resistência a antibióticos) e completar o volume com água. </li></ol></li><li> Incubar os tubos num ciclizador térmico a 50-55 ° C durante 30-60 minutos. Substituir no gelo e se transformar em E. coli através de choque térmico células quimicamente competentes. Não use pilhas electrocompetentes. A concentração elevada de sal e a concentração relativamente baixa de ADNpode fazer com que as células de arco. <ol><li> Aplicar todos os 20 ul de uma preparação comercial de 200 ul de cálcio-cloreto de E. competente coli. Incubar em gelo 30 min e choque térmico de 60 seg a 42 ° C </li><li> Retornar ao gelo 2 min, aplicar 750 ul de meio LB SOC ou recuperar e agitação num tubo de microcentrifugação de 30 minutos a 37 ° C. Placa da mistura e diluições apropriadas em LB + Kan (ou outro antibiótico apropriado). Isso pode resultar em maior fundo (e um maior número de eventos de recombinação não-alvo) do que o tradicional clonagem baseada ligadura mas vale a pena para a tela cerca de 10 colônias. </li></ol></li><li> Confirmar clone através de sequência e armazenar-se uma alíquota a -80 ° C </li></ol> 3. Biobalística transfecção de tabaco com o Gene Gun Esta é uma técnica que está estabelecida em Jove 18,19. As principais etapas e as diferenças são descritas a seguir. <ol><li> Crescer 50-100 ml E. coL ou 200 ml de Agrobacterium. A cultura-prep Maxi. Uma concentração de cerca de 1.500 ng / mL é necessário para continuar. </li><li> Prepare gene arma balas como descrito anteriormente. Coloque-os no Gene Gun. </li><li> Para a transfecção de Nicotiana benthamiana tabaco tecido foliar da epiderme, escolha uma folha grande perto da base de uma planta de 3 meses de idade. Cortá-la com cuidado e coloque-o lado de baixo para cima em papel-toalha molhada em um prato de 15 centímetros de Petri. </li><li> Ajuste a pressão Ele para o Gene Gun para 200-250 psi. </li><li> Procurar com cuidado o Gene Gun entre as nervuras no lado inferior de uma folha, a cerca de 3-5 cm acima dela. Solte a segurança e entregar as partículas para a folha. Cada bala pode ser usada duas vezes no mesmo local sobre a folha. Se as folhas explode, descartar e começar uma nova folha-haja alguma variação na dureza foliar, devido às condições de crescimento, tais como a idade, umidade, etc Para uma folha de 12 cm, à espera de encaixar cerca de 6 tiros. </li><li> Armazenar a folha, coberto com a parte superior de umPlaca de Petri para 2-3 dias com pouca luz no banco. </li><li> Examine a folha em um microscópio de dissecação equipado com fluorescência UV, e procurar por células individuais que expressam GFP. Dissecar 5-10 seções mm de folha. </li><li> Mergulhe a folha em um slide poço profundo microscópio em ~ 200 mL de 0,1% Triton X-100. O detergente ajuda a impedir que as bolhas de ar formando na superfície, e o poço de lâmina de microscópio permite uma área de imagem de tecido maiores. </li></ol> 4. Microscopia confocal do tecido Transfectadas Estas instruções variam para cada instrumento, por isso é essencial para se devidamente treinado. <ol><li> Para os experimentos de detecção de fluorescência, definir o laser de excitação a 489 nm, e definir os detectores fotomultiplicadores de 500-569 nm para fluorescência da GFP e 630-700 nm para clorofila auto-fluorescência. Células de imagem usando um objetivo de imersão em água 40x. </li></ol>

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Os autores não têm nada a revelar.

Neste estudo, as estratégias simples são descritos para a localização de uma única proteína transgénica para vários compartimentos celulares em plantas. O objetivo era projetar construção que expressasse um único gene em mais de uma organela em Nicotiana benthamiana. As estratégias incluem desenho racional de construções de DNA baseado em GFP, montagem Gibson, a entrega dos plasmídeos de células da folha com o Gene Gun, e observação dos resultados com a microscopia confocal. <p class="jove...

Este trabalho é um projeto de engenharia biologia / sintético metabólica em que as células de plantas são projetados para expressar uma proteína repórter em várias organelas, mas com apenas uma única construção de DNA. Uma abordagem para proteínas alvo de mais do que um local envolve múltiplas cópias genéticas de clonagem, contendo cada um péptido localização diferente. Cada cópia deve ser introduzido por sucessivas retransformação ou, alternativamente, por retrocruzamentos transforms solteiro 1. Isto envolve a clonagem adicional, e é limitada por uma etiqueta de localização por terminal. Outra maneira para localizar uma proteína de múltiplos locais é através de splicing alternativo 2-5. O RNA é transcrito a partir de um único gene, mas cópias diferentes da transcrição são processados ​​de maneira diferente, muitas vezes em mais do que uma maneira por célula. Isto pode resultar em mais do que um ARN mensageiro na célula transcrito a partir de um único gene. Estes messenge diferenteRNAs r pode codificar para diferentes isoformas da mesma proteína, ou no caso de uma mudança de enquadramento, uma proteína completamente diferente. Apesar de splicing alternativo foi descrito na literatura durante muitos anos, os mecanismos de acção e de doador conservada e locais de união de receptor apenas estão a ser elucidados mais recentemente 6. Como estes sites estão sendo mais bem descrito, abrem-se oportunidades para a engenharia. Engenheiros metabólicos da planta são confrontados com um desafio ao expressar uma proteína em várias organelas. Para que uma proteína que é para ser localizada a mais de um organelo, o engenheiro necessário clonar o mesmo gene várias vezes, cada uma com uma sequência de sinal separado dirigindo-a para o organelo de interesse. Para um único gene em três organelas, este é simplesmente três genes. Mas, para uma via metabólica de seis genes, esta expande-se para 18 genes, um esforço significativo clonagem. Combinando várias seqüências de localização em um sinal de um único gene, alternativamente emendadosificantly reduz esse esforço. Por exemplo, fotorrespiração reengenharia 7,8 e síntese isoprenoid 9,10 envolve tanto os cloroplastos e peroxissomos. No nosso caso, se aproveitou de locais de splicing como observado em um sistema de Arabidopsis thaliana naturais descrito anteriormente 6. Nós racionalmente redesenhado a sequência de mRNA deixando os locais de splicing naturais sozinho, mas colocado sequências que codificam alvos-peroxissoma-cloroplasto ou dentro dos intrões alternativamente emendados (Figura 1). A proteína expressa pode ou não ter uma etiqueta, dependendo se o pré-ARNm que codificava foi excisada como um intrão (Figuras 1g e 1h). Para mais informações sobre o projeto das construções apresentadas neste trabalho, consulte o artigo do companheiro 11. Porque este é ainda um esforço significativo clonagem, montagem Gibson, um novo método para a clonagem de arquétipos de ADN, é used na construção. O método de Gibson pode ser usado para qualquer sequência, independentemente de sítios de restrição (Figura 2) 12-14. A combinação específica de enzimas permite um único passo, a montagem isotérmico. Neste método, diversas peças de ADN de cadeia dupla linear são concebidos de tal modo que eles têm sequências sobrepostas de ~ 50 pb. A mistura de enzimas montagem Gibson digere parcialmente as partes de ADN lineares, expondo cadeias simples de seqüências homólogas. Estas sequências de cadeia simples parciais são re-recozido na mistura de reacção, resultando em um, de uma etapa, independente da sequência, de reacção rápida subclonagem livre de ligação. Este trabalho descreve um) projeto racional construções alternativamente emendados para expressão em cloroplastos de plantas, peroxissomos, e citosois, 2) sua clonagem usando o novo método sem ligadura da montagem Gibson, 3) a sua entrega nas células da folha de tabaco com o gene Gun, e 4) Os resultados mostram a segmentação organelo, como observado com a GFPe microscopia confocal.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligonucleotide primers</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td style="width:167px;">Design specifically for construct</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneBlocks</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td>500 bp oligonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ApE software</td>
			<td style="width:191px;">U Utah</td>
			<td style="width:119px;">(download)</td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MinElute kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">28004</td>
			<td>Used to purify PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep spin miniprep kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Used to prepare cloning-appropriate amounts of plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Master Mix with GC Buffer</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0532S</td>
			<td>Used to PCR-amplify gene of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibson assembly reaction mix</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">E2611L</td>
			<td>Master mix of the following 9 ingredients</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M Tris-HCl pH7.5</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:119px;">T1075</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M MgCl2</td>
			<td style="width:191px;">G Biosiences</td>
			<td style="width:119px;">82023-086</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP mix</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0192</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M DTT</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0861</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG-8000</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">19966</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:119px;">A1124,0005</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T5 exonuclease</td>
			<td style="width:191px;">Epicentre</td>
			<td style="width:119px;">T5E4111K</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">F530S</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Taq DNA ligase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0208L</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibthon.org</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>Website to simplify calculations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasmid PLUS Maxi kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">12963</td>
			<td>Used to prepare DNA for gene gun bullets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Gun system</td>
			<td style="width:191px;">BioRad</td>
			<td style="width:119px;">165-2451</td>
			<td>Includes all parts necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nicotania benthamiana</td>
			<td style="width:191px;">(n/a)</td>
			<td style="width:119px;">(n/a)</td>
			<td>Gift of Jen Sheen, MGH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:191px;">Leica</td>
			<td style="width:119px;">SP5 X MP</td>
			<td>Imaging of resultant cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deep well slides</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71561-01</td>
			<td>Used for confocal imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">A Plasmid Editor (ApE) </td>
			<td style="width:191px;">University of Utah </td>
			<td style="width:119px;"></td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gibthon:Ligation calculator</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>http://django.gibthon.org/tools/ligcalc/</td>
		</tr>
	</tbody>
</table>

transient gene expression in tobacco using gibson assembly and the gene gun

A fim de orientar uma única proteína para várias organelas subcelulares, plantas tipicamente duplicar os genes relevantes e expressar cada gene separadamente usando estratégias regulatórias complexas, incluindo promotores diferenciais e / ou seqüências de sinais. Engenheiros metabólicas e biólogos sintéticos interessadas no direccionamento enzimas para um organelo particular, são confrontados com um desafio: Para que uma proteína que é para ser localizada a mais de um organelo, o engenheiro necessário clonar o mesmo gene várias vezes. Este trabalho apresenta uma solução para esta estratégia: o aproveitamento splicing alternativo do mRNA. Esta tecnologia aproveita de cloroplasto estabelecida e seqüências peroxissoma segmentação e as combina em um único mRNA que é alternativamente emendados. Algumas variantes de processamento são enviados para o cloroplasto, algum do peroxissoma, e outros para o citosol. Aqui o sistema é projetado para múltiplo organela alvo com splicing alternativo. Neste trabalho, a GFP era esperado para ser exprEssed no cloroplasto, citosol, peroxissoma e por uma série de racionalmente concebidos 5 &#39;tags de mRNA. Estas etiquetas têm o potencial de reduzir a quantidade de clonagem necessária quando os genes heterólogos têm de ser expressos em vários organelos subcelulares. As construções foram concebidas em trabalho anterior 11, e foram clonados utilizando a montagem de Gibson, um método de clonagem independente de ligação que não necessita de enzimas de restrição. Os plasmídeos resultantes foram introduzidos na Nicotiana benthamiana células da epiderme da folha, com um protocolo modificado do gene da arma. Finalmente, as folhas transformadas foram observadas por microscopia confocal.

O esforço do projeto foi o resultado de um planejamento significativo. Novel para este projecto é a utilização de processamento alternativo para criar uma pré-ARNm que é traduzido em proteínas diferencialmente expressas. Estas proteínas são expressas em diferentes organelos, neste caso, o cloroplasto, peroxisoma e / ou citoplasma. Foram adaptados gene Arabidopsis natural que é alternativamente emendados 6, e colocadas conhecido cloroplasto 6 e 17 sequências de direcci...

Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Este trabalho descreve um novo método para alvejar seletivamente organelas subcelulares nas plantas, analisadas usando o BioRad Gene Gun.

Expressão Gênica transiente em tabaco usando Assembleia Gibson eo Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

transient-gene-expression-tobacco-using-gibson-assembly-gene

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20.4K visualizações.  Harvard University.  Este trabalho descreve um novo método para alvejar seletivamente organelas subcelulares nas plantas, analisadas usando o BioRad Gene Gun. 

Vídeo: Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Determinação da Microbial Extracelular Enzyme actividade nas águas, solos e sedimentos, utilizando alta taxa de transferência de microplaca Ensaios

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Ambiente

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

Transcrição e Metabolite Profiling de Avaliação dos Árvore Tabaco e Poplar como matéria-prima para a indústria baseada em Bio

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Rastreamento contaminação microbiana em ambientes de varejo com fontes fluorescentes Powder - Um ambiente de exemplo Retail Delicatessen

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expressão da proteína recombinante a partir da celulase Cel5A<em&gt; Trichoderma reesei</em&gt; Em Plantas de tabaco

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Triagem géneros alimentícios destinados a uma classe integrons e Gene Cassetes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

Controle mediado por RNAi de aflatoxinas em amendoim: método para analisar a produção de micotoxinas e Transgene Expression na Amendoim /<em&gt; Aspergillus</em&gt; Patossistema

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

A craca Balanus improvisus como modelo Marine - cultivo e expressão gênica

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium tumefaciens e Agrobacterium rhizogenes-transformação mediada de batata e a atividade de promotor de um Gene de suberina por GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

Ensaios foliares destacados para simplificar estudos de expressão gênica em batata durante a infestação por mastigação de insetos Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Estimativa do índice de área foliar usando três métodos distintos em stands deciduous puros

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...