Gracias a Michaela Hall, Larissa Bispo y Gustavo Tavares para la asistencia técnica.

Los productos alimenticios que se consumen crudos o ligeramente cocidos son de mayor preocupación para la salud humana. Los ejemplos incluyen ensalada de verduras, frutas, mariscos y crustáceos. 1. Recogida de muestras <ol><li> Recoger las muestras en condiciones que reduzcan al mínimo la contaminación, y se almacena en sacos limpios, separados durante el transporte. Una vez recogidos, las muestras deben almacenarse a 4 ° C y procesadas dentro de 24 horas. </li></ol> 2. Preparación Enriquecido Cultura <ol><li> Frutas y vegetales: <ol><li> Poner unos 10 g de material en una bolsa de plástico resistente. Si el procesamiento de material más grande, se concentran en la piel de la fruta o verdura. Añadir 90 ml 0,1 M tampón fosfato de sodio, pH 7,0 y el proceso en un mezclador de paletas durante 30 seg. Alternativamente, el material se puede agitar manualmente si un mezclador de paleta no está disponible. </li><li> Recoger el líquido en dos 50 ml tubos Falcon. Tubos centrífuga a 4.000 xg durante 7 minutos a las bacterias de pellets. </li&gt; <li> Retirar con cuidado el sobrenadante y suspender las bacterias sedimentadas en 30 ml de caldo estéril Luria Bertani (LB). </li><li> Incubar en un agitador O / N, la celebración de un tubo a 25 ° C y el otro a 37 ° C. Los cultivos deben agitaron a 200 oscilaciones por minuto (OPM). Culturas Almacenar a 4 ° C hasta su utilización. </li></ol></li><li> Mariscos: <ol><li> Diseccionar el estómago (ostras) o del tracto digestivo (gambas) con unas pinzas y pinzas estériles y colocarlos en un tubo de 1,5 ml estéril que contiene 200 l de tampón de fosfato de sodio 0,1 M, pH 7,0. Macerar la muestra para crear un homogeneizado. </li><li> Dispense 5 ml de caldo LB en dos tubos de 5 ml estériles, e inocular cada una con el homogeneizado de mariscos. </li><li> Incubar en un agitador O / N, la celebración de un tubo a 25 ° C y el otro 37 ° C. Los cultivos deben agitaron a 200 opm. Culturas Almacenar a 4 ° C hasta su utilización. </li></ol></li></ol> 3. Los cultivos de cribado para integrones MgCl2 de 2,5 mM. Si es necesario, Lorenz (2011) 24 ha descrito métodos de optimización y solución de problemas de la PCR en un número anterior de esta revista. <ol><li> Preparación de ADN Hervido: <ol><li> Prescindir de 100 l de cultivo enriquecido en un 0,5 ml tubo de PCR estériles. Calentar la muestra a 99 ° C durante 10 min, utilizando un baño de agua o bloque de calor. Encajar frialdad en hielo durante 2 min. </li><li> Microcentrífuga a 14.000 xg durante 5 min para sedimentar los restos celulares. Regresar a hielo. Las muestras de ADN pueden ser congeladas a -20 ° C hasta que sea necesario, pero deben ser descongeladas en hielo. </li></ol></li><li> La detección de la clase 1 integrones utilizando PCR: NOTA: Los cultivos mixtos de la / N incubación O son examinados para detectar la clase 1 integrones utilizando el protocolo HS463a / HS464 PCR (Tabla 1). <ol><li> Descongele todos los reactivos de PCR en hielo, lejos de cualquier sou potencialRCE de contaminación de ADN. Conforman 48 l PCR Mastermix por muestra, incluidos los controles negativos (Tabla 2). Dispensar 48 l de mezcla maestra de PCR a cada tubo, usando puntas de barrera. </li><li> Añadir 2 l de ADN descongelado para tubos de PCR individuales utilizando puntas de barrera. Ejecutar programa apropiado en el termociclador de PCR. </li><li> Para evaluar los resultados de la amplificación, a electroforesis 7 l de producto de PCR en un gel de agarosa al 2% se vertió y se ejecutan en tampón TBE (90 mM TRIS-borato, EDTA 2 mM). Incluir un marcador de peso molecular, tal como una escalera de 100 pb. </li><li> Publicar mancha el gel con una mancha de ADN, y visualizar utilizando un transiluminador UV. Compruebe que no ha habido ninguna amplificación en el control negativo. Un fuerte banda en torno a 470 pb para cualquier muestra particular indica que el cultivo mixto contiene bacterias que llevan un integrón de clase 1. Colonias puras ahora se aíslan de cualquier cultura mixta que regresó una PCR positiva. </li></ol></li></ol><ol><li> Las diluciones en serie y placas de cálculo: NOTA: Para aislar colonias individuales a partir de los cultivos mixtos PCR-positivos, el cultivo mixto se diluyó en serie 10 veces en tampón de fosfato de sodio, y luego plateado para recuperar colonias individuales. <ol><li> Añadir 1 ml de cultivo mixto a 9 ml de tampón de fosfato de sodio 0,1 M, pH 7,0 y mezclar por inversión. Añadir 1 ml de la dilución a una más 9 ml de tampón de fosfato, y repetir hasta una dilución en serie de 10 -8 se alcanza. </li><li> Corre 100 l de los 10 -4 a 10 -8 diluciones en agar LB, por duplicado. Incubar las placas de O / N a la misma temperatura usada para el cultivo mixto inicial. Las placas pueden entonces ser almacenados a 4 ° C hasta que sea necesario, sin embargo es mejor para procesar las colonias individuales bastante rápidamente. </li></ol></li><li> Selección colonia individual y preparación de ADN hervida: <ol><li> Escoja colonias individuales de las placas de dilución de serie, sínando tantos tipos de colonias diferentes como sea posible, utilizando criterios como tamaño de la colonia, forma y color. Use palillos de dientes estériles para seleccionar colonias individuales de la placa de expansión. </li><li> Toque el palillo de dientes a la colonia, y luego transferir a un tubo de PCR que contiene 100 l de agua estéril. Si un gran número de colonias son de ser controlados, a continuación, las colonias se pueden preparar en una bandeja de microtitulación. Girar el palillo de dientes entre los dedos para desalojar algunas de las células en el agua. </li><li> Utilizando el mismo palillo de dientes, inocular una placa de LB con una raya de la colonia. Un gran número de colonias se pueden almacenar en placas marcadas si cada racha es de aproximadamente 1 cm de largo. Repita los pasos 1 a 3 hasta que haya seleccionado un número adecuado de aislamientos. </li><li> Incubar el LB placas O / N, a la misma temperatura que la utilizada para el cultivo enriquecido inicial. Las culturas pueden entonces ser almacenados a 4 ° C hasta su utilización. </li><li> Para la preparación de ADN a partir de las suspensiones bacterianas, siga los pasos como outlined en el apartado 3.1. Para la selección de los lisados ​​de cultivos puros, realice el HS463a / HS464 PCR como se indica en el apartado 3.2. Aislamientos que volver una PCR positiva será utilizada para la preparación de ADN genómico (artículo 5), y para otros análisis. </li></ol></li></ol> 5. Las colonias individuales de ADN genómico La extracción de integrón positivos Uso Bead Vencer a 25 <ol><li> Se inoculan 5 ml de caldo LB con una muestra del cultivo puro aislado en el apartado 4.2. Incubar O / N, agitando a la misma temperatura que se utilizó para el cultivo enriquecido inicial. </li><li> Células de pellets a 4.000 xg durante 7 minutos, a continuación, eliminar el sobrenadante. Resuspender las células sedimentadas en 1 ml de solución CLS-TC en un tubo de preparación rápida lisis Matrix E. El uso de una máquina de golpes de talón, procesar la muestra a 5,5 m / s durante 30 s. </li><li> Tubo de centrífuga a 14.000 xg durante 5 min y se recuperan 700 l de sobrenadante en un tubo de 1,5 ml estéril. </li><li> Añadir 700 l de matriz de unión diluido 1: 5 con 6 M guanidinium tiocianato y mezclar a temperatura ambiente durante 5 min. Centrifugar a 14.000 xg durante 1 min, el sobrenadante de descarte. </li><li> Añadir 800 l de etanol de lavado de sal (70% de etanol, acetato de sodio 0,1 M) y agitar hasta que el sedimento se resuspendió completamente. Lavar durante 5 min a RT, se centrifuga a 14.000 xg durante 1 min y desechar el sobrenadante. Asegúrese de que todos sobrenadante se ha eliminado, utilizando una micropipeta si es necesario, a continuación, dejar secar al aire durante 5 minutos. </li><li> Resuspender el pellet matriz en 200 l de TE (Tris-HCl 10, 1 mM EDTA, pH 7,6) pipeteando arriba y abajo. Incubar a temperatura ambiente durante 3 min. Centrifugar a 14.000 xg durante 3 min y la transferencia de 160 l de la ADN eluido en un tubo estéril. Ejecutar una alícuota de la extracción de ADN genómico en un / gel de agarosa al 2% w v para comprobar el rendimiento y la integridad de la extracción de ADN. El ADN genómico purificado se almacena a -20 ° C y descongelado en hielo cuando sea necesario para las pruebas de PCR. </li></ol> 6. PCR de diagnóstico y secuenciación de ADN NOTA: LaADN genómico preparado en la sección 5 se utiliza para todas las PCR de diagnóstico, y para confirmar el resultado positivo de la clase 1 integrones. <ol><li> Descongelar el ADN genómico en hielo, vórtice brevemente la muestra de ADN y el giro pulso en 14.000 xg durante 20 seg. Repita la PCR para el gen de la clase 1-integrón integrasa usando HS463a / HS464 (Tabla 1), lo que confirma que el aislado es positivo para la amplificación de 471 pb (Sección 3.2). </li><li> Identificación de cultivos positivos a nivel de especie se logra mediante el análisis de la pequeña subunidad del gen rRNA (16S ADNr). Amplificar el gen 16S usando los cebadores F27 / R1492 (Tabla 1). Compruebe los productos de PCR utilizando electroforesis. Todos los objetivos bacterianas deben generar un amplicón 16S de aproximadamente 1.450 pb. </li><li> Secuencia del gen amplicón ARNr 16S para determinar identidad de la especie, pero como no es probable que sean múltiples PCR positivos desde el paso 6.2, y muchos de estos positivos serán las mismas especies bacterianas, nosotros primero distinguir las diferentes especies using digestión de restricción del producto de PCR 16S. <ol><li> Digerir una alícuota de la PCR 16S con la enzima de restricción Hinf 1. Cualquier enzima que reconoce los sitios de escisión 4 pb harán, pero Hinf 1 genera buena diversidad de 16S objetivos, y es una enzima barata fiable. </li></ol></li><li> Configure una restricción 30 l digerir mezcla maestra (Tabla 2) en un tubo estéril y añadir 20 l de producto de PCR 16S un-una vez purificada. Incubar en un baño de agua 37 ° C O / N. Revise el resumen en un gel de agarosa al 2%. </li><li> Puntuación de los patrones de digestión producidos por Hinf 1. Los aislados con el mismo perfil de restricción 16S es probable que sean la misma especie. En vez de secuenciar cada producto PCR 16S, secuencia en la mayoría de los tres representantes de cada patrón de restricción Hinf 1. </li><li> Purificar los productos de PCR 16S utilizando un kit comercial. Un kit de exonucleasa de cadena sencilla se puede utilizar, pero basa columna o métodos de precipitación también funciona bien para eliminar uninccebadores orporated y los ADN de cadena sencilla. Secuencia de los amplicones 16S utilizando r910 imprimación (Tabla 1), la creación de las reacciones según lo especificado por la instalación de secuenciación. </li><li> Utilice la secuencia de ADN resultante para interrogar a la base de datos de ADN del NCBI con la función blastn ( <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ). Más del 97% de identidad de nucleótidos se suele tomar como identidad de la especie. </li></ol> 7. Mapeo y caracterización de integrones y cassettes Arrays NOTA: Clase 1 integrones que emanan de los ecosistemas dominados por el hombre es probable que sean los integrones más comunes en todas sus muestras. Estos integrones todos tienen un solo origen reciente, por lo que tienen una secuencia de ADN altamente conservadas 5. <ol><li> Diferenciar entre una clase 1 integrones que emanan de fuentes humanas y los que se encuentran naturalmente en muestras ambientales mediante la realización de unPCR con el cebador establece intI1F165 / intI1R476 10 (Tabla 1). NOTA: Clase 1 integrones de bacterias comensales clínicos o generarán un producto ~ 300 pb, mientras que la clase del medio ambiente 1 integrones no generarán ningún producto. </li><li> Caracterizar los integrones clínicos mediante la amplificación de la matriz de cassette con el juego de primers HS458 / HS459. Esta PCR se amplificar la región entre intI1 y el segmento conservado el 3 &#39;en el extremo de la mayoría de casete arrays. Debido a que el número y la identidad de cassettes es variable, el tamaño del producto de PCR también variará. </li><li> Purificar amplicones y secuenciar el ADN utilizando cada uno de los cebadores de amplificación. Utilice secuencias para interrogar las bases de datos de ADN utilizando la función blastn ( <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ). </li><li> Asegúrese de nombrar de casetes siga nomenclatura estandarizada 9, ya que muchas deposiciones de datos para casete génico integróns usan nombres inconsistentes o ambiguas. Preste especial atención a los nuevos casetes de genes, ya que estos pueden codificar nuevos fenotipos, incluyendo genes que confieren un aumento transmisibilidad, patogenicidad o virulencia 8,21. Todavía circulan preclínicos sobre las formas de la clase 1 integrón en ambientes naturales: NOTA. En particular, aquellos que están vinculados a activos Tn 402 transposones son de mayor interés 26. Las matrices de cassette de estos integrones no amplificarán con el juego de primers HS458 / HS459, pero pueden ser amplificado con la MRG284 / MRG285 imprimación conjunto (Tabla 1), la generación de amplicones de dependiente en el contenido de cassette de tamaño variable. Productos de PCR deben ser purificadas y ADN secuenciado como se indica en 7.2. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

Los autores no tienen nada relevante a revelar.

La identificación de los integrones y sus cassettes genéticos asociados es potencialmente un paso clave en la predicción de la aparición de nuevos agentes patógenos oportunistas, el seguimiento de las vías para los patógenos en la cadena alimentaria humana, y la identificación de una nueva resistencia y virulencia determinantes 8,21,26. El objetivo de este trabajo fue describir un enfoque simplificado para el cribado de muestras para la clase 1 integrones, caracterizando sus matrices de cassette y la ...

El descubrimiento de los antibióticos fue uno de los mayores logros científicos del siglo 20. Sin embargo, el uso y abuso de antibióticos ha llevado a la rápida evolución de las bacterias resistentes a los antibióticos, y estos ahora representan una seria amenaza para la salud pública en el siglo 21. El surgimiento de cepas bacterianas resistentes a la mayoría de las opciones de tratamiento plantea la posibilidad de que estamos entrando en una era en que los medicamentos antimicrobianos ya no son eficaces 1,2. La maquinaria genética que confiere resistencia a los antibióticos es un sistema antiguo, anterior a los seres humanos y las presiones de selección de antibióticos por millones de años 3. Elementos genéticos móviles, tales como plásmidos, transposones, islas genómicas, elementos y integrones conjugativos integradores pueden difundir los genes de resistencia a antibióticos (ARG), tanto dentro como entre las especies bacterianas 4. De éstos, los integrones han jugado un papel central en la propagación de ARG, a pesar de lahecho de que se basan en plásmidos y transposones para la movilización y la inserción en los genomas bacterianos 5. Los integrones capturan casetes de genes usando un integrón-integrasa, y luego expresan casetes utilizando un integrón codificado promotor 6,7 (Figura 1). Integrón gen casetes son pequeños elementos móviles que consisten en cuadros individuales abiertos de lectura (ORF), cuyos productos pueden conferir resistencia a los antibióticos o desinfectantes 8. 1 integrones de clase son los integrones más comúnmente recuperados de aislados clínicos 5, donde han adquirido colectivamente más de 130 diferentes casetes de genes de resistencia a antibióticos 9. La difusión de la clase 1 integrones en comensales y patógenos bacterias humanas asociadas genera corrientes de desechos humanos que contienen un gran número de estos elementos genéticos 10. Se estima que 10 19 bacterias que contienen integrones de clase 1 se liberan a través de los lodos de depuradora cada año in el Reino Unido 11. Por lo tanto, no es sorprendente que la clase 1 integrones que confieren resistencias a antibióticos están siendo detectados en la microbiota de las aves silvestres, peces y otra fauna nativa 12-14. Liberar integrones de nuevo en el medio ambiente plantea un importante problema de salud pública, ya que la adquisición de nuevos cassettes genéticos y reordenamientos complejos con otros elementos móviles persiste, especialmente en las plantas de tratamiento de aguas residuales y otros cuerpos de agua 15-18. El entorno natural, entonces se convierte en un campo de reclutamiento fértil para nuevos determinantes de resistencia y patógenos oportunistas 19,20. Bacterias que contiene integrón-Novel y nuevas ARGs pueden rodear de nuevo en la comunidad humana a través del agua y alimentos contaminados 21,22. Vigilancia de ARGs ambientales es una estrategia clave para la comprensión y el manejo de la resistencia a los antibióticos en el futuro 23. En particular, se debe prestar atención a los alimentos que se comen crudos oligeramente cocidos, ya que estos presentan la mayor amenaza para la transmisión de nuevos elementos móviles y agentes patógenos. En este protocolo, un enfoque simplificado para la detección, identificación y caracterización de integrones de clase 1 y de sus cassettes genéticos asociados en los productos alimenticios se indica (Figura 2). Usando una combinación de cultivo y reacción en cadena de polimerasa (PCR), integrones se pueden detectar rápidamente en comunidades bacterianas complejas y aislamientos individuales. Se dan métodos para la identificación de las especies de bacterias y la conformación y la identidad de los casetes de genes integrón-asociado. El método es adecuado para una amplia variedad de alimentos vegetales y animales, y los ejemplos de los flujos de trabajo típicos se indican para cada uno de estos tipos de alimentos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>GoTaq Colourless Mastermix</td>
			<td>Promega</td>
			<td>M7132</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>RNAse (Ribonuclease A from bovine pancreas)</td>
			<td>Sigma</td>
			<td>R6513-10MG</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>HinFI restriction enzyme</td>
			<td>Promega</td>
			<td>R6201</td>
			<td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>GE Healthcare</td>
			<td>27400701</td>
			<td>Used as a size standard on all agarose gels</td>
		</tr>
		<tr>
			<td>GelRed DNA stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate</td>
			<td>Life Technologies</td>
			<td>AM9422</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>CLS-TC Solution</td>
			<td>MP Biomedicals</td>
			<td>6540409</td>
			<td>Resuspension solution used at the begining of the genomic DNA extraction</td>
		</tr>
		<tr>
			<td>Lysing Matrix E FastPrep tubes</td>
			<td>MP Biomedicals</td>
			<td>116914500</td>
			<td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td>
		</tr>
		<tr>
			<td>binding matrix</td>
			<td>MP Biomedicals</td>
			<td>116540408</td>
			<td>Diluted 1:5 with 6M guanidinium thiocyanate and used in the genomic DNA extraction method.</td>
		</tr>
		<tr>
			<td>Fast Prep machine</td>
			<td>MP Biomedicals</td>
			<td>Number of options available</td>
			<td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information</td>
		</tr>
	</tbody>
</table>

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.

Proyección de cultivos mixtos y aislamientos bacterianos para intI1 Conjunto de cebadores HS463a / HS464 PCR se puede utilizar para detectar la presencia del gen de la integrasa integrón de clase 1, intI1 (Figura 1). Este conjunto de cebadores funciona bien para detectar intI1 en cultivos mixtos, y también se utiliza para detectar colonias de bacterias recogidas de placas separadas (Figura 2). Aislamientos positivos deberían generar una so...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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

Screening productos alimenticios para la Clase 1 integrones y 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 ...

screening-foodstuffs-for-class-1-integrons-and-gene-cassettes

Research

JoVE Journal

Environment

9.0K vistas.  Macquarie University. This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs. 

Video: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Expresión génica transitoria en tabaco utilizando Asamblea Gibson y la pistola de genes

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

Investigación

Medio ambiente

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Semi-Alto rendimiento para Screening Potencial Sequía-tolerancia en lechuga (<em&gt; Lactuca sativa</em&gt;) colecciones de germoplasma

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

Selección de alto rendimiento de las Variaciones obstinación en lignocelulósica Biomasa: Total lignina, lignina Monómeros y enzimática Azúcar de lanzamiento

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

El ensayo de los comportamientos de alimentación depredadores en<em&gt; Pristionchus</em&gt; Y otros nematodos

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

La detección de la actividad endocrina en Uso de Agua comercialmente disponibles<em&gt; In Vitro</em&gt; Transactivación bioensayos

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocolo para microplásticos muestreo en la superficie del mar y Análisis de las muestras

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extracción de plaguicidas organoclorados de pellets de plástico y análisis de tipos de plástico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Extracción y caracterización de los agentes tensioactivos de los aerosoles atmosféricos

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

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.

El percebe Balanus improvisus como modelo Marina - cultivo y expresión del gen

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.

Tumefaciens de la agrobacteria y rhizogenes de la agrobacteria-transformación mediada de la patata y la actividad de promotor de un Gene del Suberin 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 ...