Me gustaría reconocer Olga de Souza Silva de asistencia técnica. Este trabajo fue apoyado por el Fondo Nacional Suizo (FNS) Grant 31003A_127029.

El método TransFLP (Figura 1) se ejemplifica mediante tres enfoques diferentes: i) la deleción de dos genes adyacentes que codifican los determinantes de virulencia de V. cholerae (por ejemplo, ΔctxAB), II) la extracción de una isla de patogenicidad (por ejemplo, ΔVPI-1), y III) la integración de una secuencia de T7 RNA polimerasa dependiente de promotor aguas arriba de un gen de interés, lo que permite posteriormente su artificial expresión en la cepa de fondo respectivo (por ejemplo, T7-tfox) (Figura 2). 1. Preparación de Copos de quitina <ol><li> Peso 50-80 mg de copos de quitina en un tubo de plástico estándar de 1,5 ml. Este puede ser preparado en grandes cantidades. Copos de quitina están disponibles comercialmente de Sigma (número cat. C9213). </li><li> Autoclave los copos de mantenimiento de las tapas de los tubos abiertos. </li><li> Inmediatamente después de cerrar las tapas de la autoclave se ha enfriado. </li><li> Tienda autoclave quitina escamasa temperatura ambiente. </li></ol> 2. Opcional: Transformación artificial de V. cholerae con Flp recombinasa plásmido que codifica <ol><li> Preparar electrocompetentes V. cholerae células utilizando métodos estándar 11. La tienda de alícuotas de células competentes a -80 ° C. </li><li> Añadir 1-2 l de una normal de mini-preparación de plásmido pBR-flp 5 a la electrocompetentes V. células cholerae y transferir la mezcla en una cubeta de electroporación (0,2 cm ancho hueco). </li><li> Aplicar un pulso a 1,6 kV. </li><li> Añadir 0,9 ml de medio SOC, suavemente la transferencia de células a un tubo estándar de 14 ml. Incubar que no se mueve durante 2,5 a 3 horas a 30 ° C. </li><li> Placa de 100 l y 300 l en placas de LB que contenían 100 ug / ml de ampicilina y se incuba a 30 ° C durante la noche. </li><li> Purificar solo clon y almacena como reserva de glicerol para experimentos TransFLP futuras. </li></ol> 3. Diseño de oligonucleótidos y reacción en cadena de la polimerasa <ol><li> Al menos seis oligonucleótidos se requieren para amplificar la región de ADN (s) de interés, así como el casete FRT-flanqueado antibiótico por PCR (Figura 3). Se recomienda incluir también un par de oligonucleótidos de cebado fuera del fragmento de PCR insertado. Esto permite la comprobación de la integración y la escisión correcta de la casete de resistencia a FRT-flanqueado antibiótico (por ejemplo, representado como &#39;chk-up&#39; y cebadores &#39;chk hacia abajo &quot;en las figuras 3 a 5). </li><li> Diseñar los oligonucleótidos # 2, # 3, # 4, # 5 y con cuidado. Ellos deben ser capaces de hibridarse suficientemente (por ejemplo, ~ 28 pb) a la plantilla de ADN. La plantilla de ADN es ADN genómico (gDNA) para los cebadores # 2 y # 5 y FRT-Kan-FRT-que contiene ADN plásmido tal como pROD17 12 y pBR-FRT-KAN FRT-(este estudio) para los cebadores # 3 y # 4 ( Figura 3A). </li><li> Diseño de los cebadores # 2 al # 5 que permite una amplia base-apareamiento en su extremo 5 &#39;entre el # 2 / # 3 y # 4 / # 5, respectivamente (Figura 3A </strong&gt; recuadro). Dos rondas de PCR seguir. </li><li> Realizar tres reacciones de PCR independientes como 1 ª ronda de PCR. El primero será amplificar la región corriente arriba del gen / ADN de la región de interés con la ayuda de oligonucleótidos # 1 y # 2. La PCR debería resultar en un fragmento de al menos 500 pb de longitud para permitir la recombinación homóloga dentro de las células. Fragmentos más cortos (~ 250 pb) puede ser suficiente 8 pero no se recomiendan. </li><li> En paralelo realizar la segunda PCR, que amplifica el casete FRT-flanqueado antibiótico. Para los ejemplos que aquí se utiliza principalmente aph (kan R). Uso oligonucleótidos # 3 y # 4 para esta reacción (Figura 3A). </li><li> Al mismo tiempo, amplificar la región de ADN corriente abajo por PCR (tercera muestra). El fragmento de PCR también debe ser de al menos 500 pb de longitud. Realizar la PCR con gDNA como molde y los oligonucleótidos # 5 y # 6 (Figura 3A). </li><li> Después de la purificación de todos PCR tresfragmentos, lleve a cabo la 2 ª ronda de PCR. Usar una mezcla igual de los tres fragmentos obtenidos en la primera ronda como molde. La amplificación es catalizada por los oligonucleótidos # 1 y # 6. El fragmento resultante de PCR (Figura 3B) sirve como la transformación de ADN en el experimento de transformación natural (Figura 1). </li></ol> 4. La quitina inducida por transformación natural <ol><li> Crecer V. células cholerae aeróbicamente en medio rico a 30 º C hasta alcanzar una densidad óptica a 600 nm de aproximadamente 0,5. </li><li> Cosechar las bacterias por centrifugación. Lavar la pella una vez en medio artificial definido (DASW 6) antes de resuspender las células en 2 volúmenes de DASW. Añadir 1 ml de cultivo a 50-80 mg de escamas estéril quitina (como se explica en el punto 1). Si las bacterias puerto plásmido pBR-flp (opcional punto 2), añadir ampicilina (50 mg / ml) a la cultura. Incubar a 30 ° C durante 16-24 h sin movimiento. </li><li> Añadir &amp; ge; 200 ng de la 2 ª ronda de PCR fragmento derivado (que se explica en el punto 3). Mezclar cuidadosamente sin ampliamente separar las bacterias de las superficies de quitina. Incubar a 30 ° C durante 24 horas sin movimiento. </li><li> Vortex la cultura extensamente durante ≥ 30 segundos. Corre 100-300 l selectivos en placas de LB medio (por ejemplo, kanamicina o gentamicina que contienen placas de LB ejemplos proporcionados aquí). El uso de dos placas selectivas si las bacterias albergar el plásmido pBR-flp añadiendo ampicilina concomitantemente a los otros antibióticos. Incubar las placas a 30 ° C durante 16-24 horas o hasta que las colonias son visibles. </li><li> Aislar transformantes individuales de placas selectivas. Tales transformantes han sustituido el locus cromosómico original por el fragmento de PCR debido a un evento de doble entrecruzamiento. Estas cepas se pueden usar directamente para experimentos adicionales en caso de que la eliminación de la casete de antibiótico no es necesario. </li></ol> 5. La eliminación selectiva de casete (s) por Flp-Recombinación <ol><li> Artificialmente transformar sus transformantes con el plásmido pBR-flp como se describe en el punto 2 si no se hace antes de que el ensayo de transformación natural. </li><li> Cultivar las bacterias en placas de agar LB que contenían ampicilina a 37 ° C durante 16-24 h. Opciones: cambiar la temperatura entre el 2-3 hr a 40 ° C, según la expresión de FLP del plásmido pBR-flp se de-reprimidos a altas temperaturas; 5,10 bacterias de transferencia a placas frescas después de aprox. 8 horas de incubación. </li><li> Prueba de sensibilidad a los antibióticos (demostrada aquí para kanamicina; Figura 1) por restreaking los clones en paralelo sobre placas de agar que contienen antibiótico ni antibióticos. Aislar colonias individuales sensibles. Congelar clon resistente a la ampicilina como la acción del glicerol si tiene intención de modificar aún más la tensión con supresión otro / inserción con TransFLP. </li></ol> 6. Curado del plásmido <ol><li> Cultive hasta el día bajo condiciones aeróbicas ya 30 º C. Usar medio rico sin la adición de ningún antibiótico. </li><li> Opcional: Crecer cultivo fresco por 3-6 horas por dilución de 1:100 del cultivo de una noche fresca en medio libre de antibióticos. </li><li> Placa o estría de dilución (s) de cultivo en placa de agar LB sin formato (s) e incubar a 30 ° C durante 8-16 h (hasta que las colonias son visibles). </li><li> Prueba de sensibilidad a la ampicilina de los clones por restreaking los clones en paralelo sobre placas de agar que contienen antibióticos y libre de antibióticos-(Figura 1). Congelar ampicilina cepa sensible como la acción del glicerol. </li></ol>

<ol>
	<li>Miller, V. L., Mekalanos, J. 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+novel+suicide+vector+and+its+use+in+construction+of+insertion+mutations:+osmoregulation+of+outer+membrane+proteins+and+virulence+determinants+in+Vibrio+cholerae+requires+toxR.">A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR.</a> J. Bacteriol. 170, 2575-2583 (1988).</li><li>Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., Kushner, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+generating+deletions+and+gene+replacements+in+Escherichia+coli.">New method for generating deletions and gene replacements in Escherichia coli.</a> J. Bacteriol. 171, 4617-4622 (1989).</li><li>Kato, C., Ohmiya, R., Mizuno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+disrupting+genes+in+the+Escherichia+coli+genome.">A rapid method for disrupting genes in the Escherichia coli genome.</a> Biosci. Biotechnol. Biochem. 62, 1826-1829 (1998).</li><li>Donnenberg, M. S., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+eae+deletion+mutant+of+enteropathogenic+Escherichia+coli+by+using+a+positive-selection+suicide+vector.">Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.</a> Infect. Immun. 59, 4310-4317 (1991).</li><li>De Souza Silva, O., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+Vibrio+cholerae+by+combining+natural+transformation+with+FLP+recombination.">Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination.</a> Plasmid. 64, 186-195 (2010).</li><li>Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. -. Y., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+induces+natural+competence+in+Vibrio+cholerae.">Chitin induces natural competence in Vibrio cholerae.</a> Science. 310, 1824-1827 (2005).</li><li>Pollack-Berti, A., Wollenberg, M. S., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+fischeri+requires+tfoX+and+tfoY.">Natural transformation of Vibrio fischeri requires tfoX and tfoY.</a> Environ. Microbiol. 12, 2302-2311 (2010).</li><li>Marvig, R. L., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+cholerae+as+a+tool-optimizing+the+procedure.">Natural transformation of Vibrio cholerae as a tool-optimizing the procedure.</a> BMC Microbiol. 10, 155 (2010).</li><li>Blokesch, M., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serogroup+Conversion+of+Vibrio+cholerae+in+Aquatic+Reservoirs.">Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.</a> PLoS Pathog. 3, e81 (2007).</li><li>Cherepanov, P. P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+in+Escherichia+coli:+TcR+and+KmR+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158, 9-14 (1995).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T., Ford, N., Nolan, C., Ferguson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. 1, (1989).</li><li>Reyes-Lamothe, R., Possoz, C., Danilova, O., Sherratt, 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=Independent+positioning+and+action+of+Escherichia+coli+replisomes+in+live+cells.">Independent positioning and action of Escherichia coli replisomes in live cells.</a> Cell. 133, 90-102 (2008).</li><li>Karaolis, D. K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vibrio+cholerae+pathogenicity+island+associated+with+epidemic+and+pandemic+strains.">A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.</a> Proc. Natl. Acad. Sci. USA. 95, 3134-3139 (1998).</li><li>Ikeda, R. A., Ligman, C. M., Warshamana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T7+promoter+contacts+essential+for+promoter+activity+in+vivo.">T7 promoter contacts essential for promoter activity in vivo.</a> Nucleic Acids Res. 20, 2517-2524 (1992).</li><li>Pan, S. H., Malcolm, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+background+expression+and+improved+plasmid+stability+with+pET+vectors+in+BL21+(DE3).">Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).</a> Biotechniques. 29, 1234-1238 (2000).</li><li>Faruque, S. M., Zhu, J., Asadulghani, M., Kamruzzaman, J. J., Mekalanos, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+diverse+toxin-coregulated+pilus-positive+Vibrio+cholerae+strains+fails+to+demonstrate+evidence+for+Vibrio+pathogenicity+island+phage.">Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage.</a> Infect. Immun. 71, 2993-2999 (2003).</li><li>Joelsson, A., Liu, Z., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+phenotypic+diversity+of+quorum-sensing+systems+in+clinical+and+environmental+isolates+of+Vibrio+cholerae.">Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae.</a> Infect. Immun. 74, 1141-1147 (2006).</li><li>Heidelberg, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequence+of+both+chromosomes+of+the+cholera+pathogen+Vibrio+cholerae.">DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.</a> Nature. 406, 477-483 (2000).</li></ol>

No hay conflictos de interés declarado.

El método TransFLP descrito anteriormente y en otros lugares 5 ha sido ampliamente utilizado en nuestro laboratorio. El tipo de manipulaciones genéticas que son factibles incluyen, entre otros: la supresión de los genes individuales y grupos de genes, la supresión de las islas genómicas (por ejemplo, VPI-1), la inserción de secuencias de aguas arriba de un gen de interés (por ejemplo, secuencias de promotor) y la inserción de secuencias en el extremo de un gen específico (por ej...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> La quitina escamas </td><td> Sigma </td><td> C9213 </td><td> Autoclave necesario </td></tr><tr><td> OPCIONAL: Micropulser (en caso de casete antibiótico debe ser extirpado por recombinasa Flp codificada en pBR-flp) </td><td> Biorad </td><td> 165-2100 </td><td> O electroporators comparables </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

Hay varios métodos disponibles para manipular los cromosomas bacterianos 1-3. La mayoría de estos protocolos se basan en la inserción de plásmidos replicativos condicionalmente (por ejemplo, albergar replicones pir-dependientes o sensibles a la temperatura 1,2). Estos plásmidos se integran en los cromosomas bacterianos basado en la recombinación mediada por homología. Tales mutantes de inserción son a menudo utilizados directamente en la configuración experimental. Alternativamente, la selección para la escisión del plásmido seguido por su pérdida puede ser realizado, para que las bacterias Gram negativas a menudo se basa en la enzima sacarasa contraseleccionable levano codificada por el gen sacB 4. La escisión puede restaurar el genotipo pre-inserción o resultado en un intercambio entre el cromosoma y la copia codificada por plásmidos del gen modificado. Una desventaja de esta técnica es que requiere mucho tiempo. El plásmido tiene que ser clonado primero, que requiere la transferencia horizontal en V. cholerae más (n<html> otably en el apareamiento con una E. coli cepa donante) o transformación artificial de este último, y la escisión del plásmido es aleatoria y puede restaurar el genotipo inicial o crear la modificación deseada si no hay selección positiva se ejerce. Aquí, se presenta un método para la manipulación rápida de la V. cholerae cromosoma (s) 5 (Figura 1). Este método se basa en TransFLP el recientemente descubierto quitina mediada por la inducción de competencia natural en este organismo representante 6 y otro del género Vibrio, como V. fischeri 7. Competencia natural permite la captación de ADN libre incluyendo fragmentos de ADN generados por PCR. Una vez absorbido, el ADN se recombina con el cromosoma dada la presencia de un mínimo de 250-500 pb de la región homóloga flanqueante 8. Incluyendo un marcador de selección en-entre estas regiones flanqueantes permite la fácil detección de los transformantes que ocurren con frecuencia. t &quot;&gt; Este método se puede utilizar para diferentes manipulaciones genéticas de V. cholerae y bacterias naturalmente competentes también potencialmente otros. Proporcionamos tres ejemplos novedosos en lo que puede lograrse por este método, además de nuestro estudio publicado anteriormente sobre las supresiones de genes individuales y el Además de las secuencias de etiqueta de afinidad-5. Varios pasos de optimización sobre el protocolo inicial de quitina inducida por transformación natural 6 se incorporan en este protocolo TransFLP. Estos incluyen entre otros la sustitución de fragmentos de conchas de cangrejo por comercialmente disponible quitina copos 8, la donación de PCR derivado de ADN como la transformación de material 9, y la adición de sitios diana de recombinación FLP-(FRT) 5. sitios FRT permitir la escisión dirigida a sitio del marcador de selección mediada por la recombinasa Flp 10.

Los resultados representativos de los tres ejemplos que se muestran en las Figuras 4 a 6. El primer enfoque dirigido a eliminar los genes vecinos ctxA y ctxB. Juntos codifican el factor de virulencia de V. cholerae, la toxina del cólera. Hemos diseñado oligonucleótidos para el método TransFLP como se ha descrito anteriormente y según la figura 3. La cepa parental, la cepa intermedia albergar el casete FRT-flanqueado resistencia a los antibióticos en el l...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

Un método rápido para modificar el genoma de<em&gt; V. cholerae</em&gt; Se describe. Estas modificaciones incluyen la supresión de los genes individuales, grupos de genes y las islas genómicas, así como la integración de secuencias cortas (por ejemplo, elementos promotores o secuencias de etiqueta de afinidad-). El método se basa en la transformación natural y recombinación FLP-.

TransFLP - Un método para modificar genéticamente<em&gt; Vibrio cholerae</em&gt; Sobre la base de la transformación natural y la recombinación FLP

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Research

JoVE Journal

Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K vistas.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Un método rápido para modificar el genoma de V. cholerae Se describe. Estas modificaciones incluyen la supresión de los genes individuales, grupos de genes y las islas genómicas, así como la integración de secuencias cortas (por ejemplo, elementos promotores o secuencias de etiqueta de afinidad-). El método se basa en la transformación natural y recombinación FLP-.

Video: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonización de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Investigación

Inmunología e Infección

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

A Parasite Rescate y Transformación de ensayo para la detección antileishmanial contra intracelular<em&gt; Leishmania donovani</em&gt; Amastigotes en THP1 Humanos aguda línea celular de leucemia monocítica

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicación clínica de<em&gt; La Bella Durmiente</em&gt; Artificial y células presentadoras de antígenos para modificar genéticamente las células T de la sangre del cordón umbilical y periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementación de un Sistema de infección basada en la inserción de membrana permeable para estudiar los efectos de toxinas secretadas bacterianas en células huésped de mamífero

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desarrollo de una más sensible y específica cromogénico medio de agar para la detección de<em&gt; Vibrio parahaemolyticus</em&gt; Y Otros<em&gt; Vibrio</em&gt; Especies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Una plataforma de ensayos anti-biopelícula Adecuado para la Exploración de bibliotecas de compuestos naturales

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

La neuraminidasa Ensayo de inhibición basado en la fluorescencia para evaluar la susceptibilidad de los virus de la influenza a El Inhibidor de la neuraminidasa clase de antivirales

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Técnicas de laboratorio utilizadas para mantener y diferenciar los biotipos de<em&gt; Vibrio cholerae</em&gt; Aislados Clínicos y Ambientales

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...