Name: producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes
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Se obtuvieron cepas de colección de Keio del proyecto nacional de BioResource (NIG, Japón): e. coli. Agradecemos a Dirk Linke (Departamento de ciencias biológicas, Universidad de Oslo) por su continuo apoyo. Este trabajo fue financiado por el Consejo de investigación de Noruega joven investigador subsidio 249793 (Jack C. Leo).

1. las cepas y plásmidos
<ol><li>
Cepas bacterianas
	<ol>
<li>Uso de la e. coli cepas BW251135, JW4179 (BW25113 tamA::kan)7, BL21(DE3)20y BL21ΔABCF21. Para más información ver la Tabla de materiales .</li>
	</ol>
</li>
	<li>
Bacteriófagos
	<ol>
<li>Utilice el fago P1vir para la transducción general. Almacenar el phage como un líquido con unas gotas de cloroformo (ver paso 2.2). Para obtener más información, vea Tabla de materiales.</li>
	</ol>
</li>
	<li>
Plásmidos
	<ol>
<li>Utilice el siguiente plásmidos en este protocolo: pCP2022, pIBA2-YadA23y pEibD1024. Plásmidos de control, utilizar IBA2 pASK y pET22b (véase Tabla de materiales).</li>
	</ol>
</li>
	<li>
Condiciones de crecimiento
	<ol>
<li>Propagar las bacterias en un medio25 de lisogenia caldo (LB) con vigoroso (180 – 200 rpm) a 37 ° C o 30 ° C en el caso de las cepas que contienen pCP20 y BL21ΔABCF.</li>
		<li>Realizar plásmido curado a 43 ° C.</li>
		<li>De un medio sólido, suplemento LB con agar al 1% (p/v).</li>
		<li>Para top agar, suplemento LB con 0,7% de agar y 10 mM de CaCl2 y autoclave el medio. Medio SOC de uso para la recuperación después de la electroporación26.</li>
		<li>Utilizar las siguientes concentraciones de antibióticos: 100 μg/mL de Amp y 25 μg/mL para el Kan.</li>
	</ol>
</li>
</ol>2. preparación de un lisado de fagos
<ol><li>
Infección de la cepa donante
	<ol>
<li>Crecer la cepa donante JW4197 en 5 mL de medio LB suplementada con 10 mM de CaCl2 y opcionalmente con Kan (25 μg/mL) a una densidad óptica a 600 nm (OD600) de ~ 1.0. Mide el valor de OD600 usando un espectrofotómetro.</li>
		<li>Hacer una serie de diluciones de un stock de fagos P1 existente en el medio LB: recomienda diluciones son entre 10-3 a 10-7.</li>
		<li>Mezcla 200 μL de la suspensión bacteriana y 100 μL de una dilución de phage dado en un tubo de centrífuga de 15 mL o equivalente. Preparar tantos tubos como diluciones de fago. Incubar los tubos durante 20 min a 37 ° C sin agitación.</li>
		<li>Añadir 3 mL de agar superior fundida (~ 50 ° C) suplementado con 10 mM de CaCl2 en los tubos, homogeneizar el contenido con un vórtex los tubos poco y vierta la mezcla en placas LB precalentadas para hacer capas incluso.</li>
		<li>Incubar las placas durante la noche a 37 ° C.</li>
	</ol>
</li>
	<li>
Preparación de lisado
	<ol>
<li>Al día siguiente elegir un plato con un semi-confluente crecimiento de placas de fagos. En un plato semi confluente, aproximadamente la mitad la superficie de la placa es evidente (figura 3).</li>
		<li>Raspar la capa de agar superior de dicha placa con un asa de inoculación o una herramienta similar y colocar el agar superior en un tubo de centrífuga. Añadir 1-2 mL de LB y una gota de cloroformo y agitar el tubo vigorosamente durante ~ 1 min agregar el cloroformo en una campana de humos.</li>
		<li>Centrifugar el tubo durante 15 min a 4.000 x g o más rápido para que sedimenten las células bacterianas y agar.</li>
		<li>Pasar el sobrenadante a un tubo de microcentrífuga fresco, evitando transportar cualquier residuo de la pastilla. Añadir 2 gotas de cloroformo y almacenar el lisado a 4 – 10 ° C. Añadir cloroformo en una campana de humos. No congele el phage lisado, esto resultará en una reducción significativa del número de partículas infecciosas.</li>
	</ol>
</li>
	<li>
Determinar el título del lisado
	<ol>
<li>Crecer BW25113 en LB suplementado con 10 mM de CaCl2 a 37 ° C hasta que el cultivo alcanza un OD600 de ~ 1.0.</li>
		<li>Preparar una serie de diluciones de los fagos lisado en LB (p. ej., 10-6-10-9). Nota: Tenga cuidado al pipetear la muestra desde la parte superior del lisado para evitar transferencia de cloroformo a las diluciones.</li>
		<li>Mezcla 200 μL de la suspensión bacteriana y 100 μL de una dilución de phage dado en un tubo de centrífuga de 15 mL o equivalente. Preparar tantos tubos como diluciones de fago. Incubar los tubos durante 20 min a 37 ° C sin agitación.</li>
		<li>Añadir 3 mL de agar superior fundida (~ 50 ° C) suplementado con 10 mM de CaCl2 en los tubos, homogeneizar el contenido con un vórtex los tubos poco y vierta la mezcla en placas LB precalentadas para hacer capas incluso.</li>
		<li>Incubar las placas durante la noche a 37 ° C.</li>
		<li>Al día siguiente cuenta el número de placas (regiones claras en la placa bacteriana) para cada placa y calcular el título del lisado mediante la fórmula siguiente: <img alt="Equation 1" src="/files/ftp_upload/58267/58267eq1.jpg"> Ejemplo: En las placas de las diluciones 10-710-8y 10-9, hay 231, 27 y 2 placas, respectivamente. 100 μL de cada dilución se utilizó para la infección, y porque el título se expresa como placa formando unidades (pfu) / mL, el factor de la galjanoplastia es 10. Estos números se introducen en la fórmula: <img alt="Equation 2" src="/files/ftp_upload/58267/58267eq2.jpg"> Así, el título es aproximadamente 2 x 1010 infecciosas fagos partículas/mL.</li>
	</ol>
</li>
</ol>3. P1 transducción
<ol><li>
Preparación de las células del receptoras
	<ol>
<li>Crecer la cepa receptora BL21ΔABCF en LB complementado a una densidad óptica a 600 nm (OD600) de ~ 1.0. Use un espectrofotómetro para medir el valor de OD600 .</li>
		<li>Calcular el volumen del lisado de fagos para alcanzar una multiplicidad de valor de infección (MOI) de 0,5. Para calcular el MOI, estimar el número de bacterias basado en OD600 de la cultura. Asume que un valor de600 OD de 1.0 corresponde a ~ 109 UFC/mL. Calcular el volumen requerido basado en el conocido título del lisado. Ejemplo (basado en la concentración calculada en el ejemplo después paso 2.3.6): <img alt="Equation 3" src="/files/ftp_upload/58267/58267eq3.jpg">
</li>
		<li>Añadir CaCl2 a la cultura receptora cepa a 10 mM y mezclar. Tomar 1 mL del cultivo para la transducción.</li>
	</ol>
</li>
	<li>
Realizar la transducción de señales
	<ol>
<li>Añadir el volumen adecuado de lisado de fagos a 1 mL de la cultura receptora (incluyendo CaCl2 a 10 mM) calculado en el paso 3.1.2 y mezclar suavemente. Nota: Tenga cuidado al pipetear la muestra desde la parte superior del lisado para evitar transferencia de cloroformo a la mezcla.</li>
		<li>Estáticamente, incubar la mezcla por 20 min a 37 ° C.</li>
		<li>Detener la infección mediante la adición de citrato de sodio, pH 5.5, a 100 mM.</li>
		<li>Centrifugue las bacterias (5.000 x g durante 2 min) Quite el sobrenadante y resuspender en 1 mL de LB fresco suplementado con citrato de sodio 100 mM, pH 5,5.</li>
		<li>Lavan las células dos veces más que en el paso 3.2.4 para la eliminación de calcio y phages gratis.</li>
		<li>Resuspender las bacterias en 1 mL de LB fresco suplementado con citrato de sodio 100 mM, pH 5,5. Incubar las bacterias a 30 ° C por 1 h con agitación (> 100 rpm).</li>
		<li>Recoger las bacterias por centrifugación (5.000 x g durante 2 min) y resuspender en ~ 100 μL de LB con citrato de sodio 100 mM, pH 5,5.</li>
		<li>Trasmitir las bacterias en una placa LB suplementado con Kan en 25 μg/mL y citrato de sodio 10 mM, pH 5,5 y crecer las bacterias a 30 ° C hasta que las colonias (~ 24 h).</li>
	</ol>
</li>
	<li>
Selección de las transductants
	<ol>
<li>Una vez que las colonias han crecido en la placa de selección, les restreak LB + Kan solo colonias y crecen a 30 ° C hasta que las colonias solo aparecen.</li>
	</ol>
</li>
</ol>4. suprimir el cassette de Kan
<ol><li>
La transformación con el plásmido de recombinación
	<ol>
<li>Realizar el espectro de tensión de BL21ΔABCF Kan-resistente.<ol>
<li>Crece la tensión de una sola Colonia en fresco LB (5 mL) complementado con Kan (25 μg/mL) a 30 ° C hasta que el OD600 ~0.5 – 0,7.</li>
			<li>De aquí en adelante, llevar a cabo los siguientes pasos a 4 ° C o en hielo. Centrifugue las bacterias (5.000 x g por 10 min) y eliminar el sobrenadante.</li>
			<li>Lavar el precipitado con agua destilada helada de células dos veces repitiendo el paso anterior de centrifugación y, finalmente, Resuspender el precipitado de células en 100 μl de helada glicerol al 10%.</li>
		</ol>
</li>
		<li>Enfriar cubetas de electroporación de 1 mm en el hielo.</li>
		<li>Añadir 1 pg de ADN (pCP20) plásmido a la suspensión de células, mezclar suavemente la suspensión y transferir a una cubeta de electroporación refrescado.</li>
		<li>Establecer la electroporator a 1,8 kV y electroporate las células.</li>
		<li>Rescatar las células transformadas agregando 1 mL de medio SOC y creciendo por 1 h a 30 ° C.</li>
		<li>Placa de 100 μl de las células en LB + Amp placas y crecen las células en 30 ° C durante la noche.</li>
	</ol>
</li>
	<li>
Inducción de la recombinación
	<ol>
<li>Colonias solo Pick LB + amplificador de la placa e inoculan fresco LB omitiendo todos los antibióticos.</li>
		<li>Crecen las células a la temperatura no permisiva (43 ° C) durante la noche para inducir la expresión de recombinase FLP.</li>
	</ol>
</li>
	<li>
Selección de recombinantes
	<ol>
<li>Hacer diluciones seriadas y 50 μl de una dilución de6 5-10 10 en placas no selectivo de la placa y crecer durante la noche a 30 ° C.</li>
	</ol>
</li>
</ol>5. verificación de la canceladura del Gene
<ol><li>
Verificación de la pérdida del plásmido y recombinación exitosa
	<ol>
<li>Racha solo las colonias de las placas de preparado en el paso 4.3.1 en Kan, LB, LB + Amp y las placas LB sin antibióticos, en este orden. Para ayudar en las rayas, utilice una rejilla de Colonia (véase la archivo adicional 1). Crecen las placas a 30 ° C hasta que las colonias (~ 24 h).</li>
		<li>Recoger 10-20 clones que han crecido en la placa no selectivo pero no crecen en medios selectivos para mayor verificación.</li>
	</ol>
</li>
	<li>
Verificación adicional de colony PCR
	<ol>
<li>Realizar una colonia PCR con cebadores que flanquean el tamA secuencia de codificación (véase Tabla de materiales).</li>
		<li>Preparar una mezcla PCR master. La cantidad de mezcla necesitada depende del número de colonias a ser probado (ver ejemplo en tabla 1). Mezclar los reactivos en el hielo, añadir la polimerasa de la última.</li>
		<li>Homogeneizar la mezcla PCR master y dispensar 20 μl en tubos de una tira PCR. Recoger una pequeña cantidad de cada colonia para ser proyectada utilizando una pipeta estéril y añadir a un tubo. No olvide incluir la cepa receptora original para la comparación. Opcionalmente, también incluyen la cepa donante.</li>
		<li>Ejecutar una reacción de PCR (ver tabla 2 para el programa utilizado).</li>
		<li>Preparar un gel de agarosa al 1%.<ol>
<li>Para un gel de 50 mL, medir de 0.5 g de agarosa y añadir 50 mL de buffer TAE (40 mM Tris, ácido acético al 20 mM y 1 mM EDTA, pH 8.0). Calienta la mezcla en el microondas hasta que se disuelva la agarosa todos.</li>
			<li>Una vez que la agarosa se disuelva, enfriar la solución a aproximadamente 50 ° C y luego añadir 5 μL de ADN tinción colorante. Mezcle bien la solución y verterla en una bandeja de gel en una cámara de fundición. Introduzca una o más filas de peines bien hay pozos suficientes para todas las muestras, así como marcadores de tamaño molecular. Permita que el gel para 30 minutos.</li>
		</ol>
</li>
		<li>Una vez finalizado el plazo de la polimerización en cadena, añadir 4 μL de 6 x tinte carga de ADN a cada muestra. Colocar el gel en la cámara de electroforesis y añadir Tampón TAE hasta que los pozos están cubiertos.</li>
		<li>Aplicar 10-15 μl de cada reacción de PCR a un pozo en el gel. También añadir 5 μl del marcador de tamaño molecular. A continuación, ejecute el gel durante 30 minutos a 75 V.</li>
		<li>Una vez finalizado el plazo, el gel utilizando a un sensor de luz azul de la imagen.</li>
	</ol>
</li>
</ol>6. otras técnicas
<ol><li>
Expresión de la proteína Nota: La expresión de proteínas de la prueba (YadA y EibD) ha sido descrito en detalle en otra parte23,24. Este es un breve resumen de los pasos principales.<ol>
<li>Transformar BL21ΔABCF ΔtamA with la plásmidos necesaria (YadA pIBA2 y pEibD10 y la plásmidos de control correspondiente) y seleccione transformantes en LB + Amp.</li>
		<li>Para la expresión de la proteína, inocular 100 mL de medio LB + Amp con 1 mL de un cultivo durante la noche de bacterias transformadas y crecer a 30 ° C hasta la fase de registro medio, momento en el que, inducir la producción de proteína con cualquiera de los dos anhydrotetracycline (a 100 ng/mL) o thiogalactoside de isopropilo (a 0,5 mM).</li>
		<li>Después de 2 h de la inducción a 30 ° C, medir la turbidez de los cultivos y recolectar un número de células correspondientes a 50 mL en OD600 = 1.0 por centrifugación de los cultivos durante 15 min a 4.000 x g. Lavar el sedimento 1 x con 10 mM HEPES, pH 7,4 y luego o bien almacenarlo a-20 ° C o proceso adicional como en el paso 6.2.</li>
	</ol>
</li>
	<li>
Extracción de la membrana externa Nota: La extracción de la membrana externa se realiza como se describe en detalle por León et al. 27. a continuación se resumen los pasos principales.<ol>
<li>Resuspender el precipitado de células en 1 mL de 10 mM HEPES, pH 7,4, complementado con 10 mM MgCl2 MnCl2, lisozima (0,1 mg/mL) y una pizca de ADNasa I.</li>
		<li>Lyse las células (e.g., usando un batidor de grano).</li>
		<li>Centrifugar las células poco (2 min a 15.600 x g) para eliminar restos celulares y trasladar el sobrenadante a un tubo nuevo.</li>
		<li>Centrifugar las células durante 30 min a 16.000 x g, después de lo cual, Resuspender el pellet en 400 μL de 1% N-lauroil sarcosina en 10 mM HEPES, pH 7,4.</li>
		<li>Incubar las células con agitación durante 30 min a temperatura ambiente, después de lo cual, centrifugar por 30 min como el anterior.</li>
		<li>Lave el translúcido pellet 1 x con 200 μL de 10 mM HEPES, pH 7,4, resuspender en 30 μL de 10 mM HEPES y añade 10 μL de tampón de SDS-PAGE 4.</li>
	</ol>
</li>
	<li>
Ensayos de actividad Nota: Realizar los análisis de SDS-PAGE y actividad para YadA y EibD descrito28. A continuación se resumen los pasos principales.</li>
	<li>
SDS-PAGE
	<ol>
<li>Calentar las muestras a 50 ° C durante 5 minutos antes de cargar en un gel de poliacrilamida, para evitar la desnaturalización de las proteínas.</li>
		<li>Después de la separación en SDS-PAGE, transferir las proteínas a una membrana (PVDF) de difluoruro de polivinilideno.</li>
		<li>Después de la transferencia, bloquear la membrana con 2% de polvo de leche desnatada en PBS.</li>
	</ol>
</li>
	<li>
YadA-colágeno lejos-de western blot
	<ol>
<li>Después de los bloqueos, añadir colágeno bovino tipo que diluido en solución amortiguadora de bloqueo a una concentración de 10 μg/mL e incubar la membrana para 1 h.</li>
		<li>Lavar la membrana 2 x con PBS + 0.05% Tween20 (PBS-T).</li>
		<li>Añadir el anticuerpo primario (monoclonal anti colágeno COL-1) a la membrana, 1:2,000 diluido en solución amortiguadora de bloqueo.</li>
		<li>Después de incubar la membrana durante 1 hora, lavar 2 x como se menciona en el paso 6.3.2.2 y luego añadir el anticuerpo secundario [cabra anti-ratón IgG conjugado-rábano peroxidasa (HRP)], diluido 1: 10,000 en solución amortiguadora de bloqueo.</li>
		<li>Incubar la membrana durante 1 hora, luego lavar x 2 con PBS-T. Añadir un sustrato quimioluminiscente mejorado a la membrana según las instrucciones del fabricante y detectar la banda usando una cámara CCD.</li>
	</ol>
</li>
	<li>
IgG por EibD ensayo obligatorio
	<ol>
<li>Después de los bloqueos, añadir un anticuerpo secundario (cabra anti-conejo HRP), diluido 1:2,000 en solución amortiguadora de bloqueo.</li>
		<li>Incubar la membrana durante 1 hora, luego lavar x 2 con PBS. Realizar la detección quimioluminiscente como se menciona en el paso 6.3.2.5.</li>
	</ol>
</li>
</ol>

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E., Naka, Y., Nagase, M., Mori, H., Kitakawa, 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+genome-wide+approach+to+identify+the+genes+involved+in+biofilm+formation+in+E.+coli.">A genome-wide approach to identify the genes involved in biofilm formation in E. coli.</a> DNA Research. 14 (6), 237-246 (2008).</li><li>Heinz, E., 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=Conserved+features+in+the+structure,+mechanism,+and+biogenesis+of+the+inverse+autotransporter+protein+family.">Conserved features in the structure, mechanism, and biogenesis of the inverse autotransporter protein family.</a> Genome Biology and Evolution. 8 (6), 1690-1705 (2016).</li><li>Selkrig, 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=Discovery+of+an+archetypal+protein+transport+system+in+bacterial+outer+membranes.">Discovery of an archetypal protein transport system in bacterial outer membranes.</a> Nature Structural &amp; Molecular Biology. 19 (5), 506-510 (2012).</li><li>Stubenrauch, C., 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=Effective+assembly+of+fimbriae+in+Escherichia+coli+depends+on+the+translocation+assembly+module+nanomachine.">Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine.</a> Nature Microbiology. 1 (7), 16064 (2016).</li><li>Leo, J. C., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunoglobulin-binding+Eib+proteins+from+Escherichia+coli+are+receptors+for+IgG+Fc.">The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc.</a> Molecular Immunology. 46 (8-9), 1860-1866 (2009).</li><li>M&#252;hlenkamp, M., Oberhettinger, P., Leo, J. C., Linke, D., Sch&#252;tz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+adhesin+A+(YadA)+-+Beauty+&amp;+beast.">Yersinia adhesin A (YadA) - Beauty &amp; beast.</a> International Journal of Medical Microbiology. 305 (2), 252-258 (2015).</li><li>Studier, F. W., Moffatt, 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=Use+of+bacteriophage+T7+RNA+polymerase+to+direct+selective+high-level+expression+of+cloned+genes.">Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.</a> Journal of Molecular Biology. 189 (1), 113-130 (1986).</li><li>Meuskens, I., Michalik, M., Chauhan, N., Linke, D., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strain+collection+for+improved+expression+of+outer+membrane+proteins.">A new strain collection for improved expression of outer membrane proteins.</a> Frontiers in Cellular and Infection Microbiology. 7, 464 (2017).</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 (1), 9-14 (1995).</li><li>Grosskinsky, U., 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=A+conserved+glycine+residue+of+trimeric+autotransporter+domains+plays+a+key+role+in+Yersinia+adhesin+A+autotransport.">A conserved glycine residue of trimeric autotransporter domains plays a key role in Yersinia adhesin A autotransport.</a> Journal of Bacteriology. 189 (24), 9011-9019 (2007).</li><li>Leo, J. C., 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=The+structure+of+E.+coli+IgG-binding+protein+D+suggests+a+general+model+for+bending+and+binding+in+trimeric+autotransporter+adhesins.">The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.</a> Structure. 19 (7), 1021-1030 (1993).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+lysogenesis.+I.+The+mode+of+phage+liberation+by+lysogenic+Escherichia+coli.">Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli.</a> Journal of Bacteriology. 62 (3), 293-300 (1951).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> Journal of Molecular Biology. 166 (4), 557-580 (1983).</li><li>Leo, J. C., Oberhettinger, P., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+outer+membrane+insertion+and+folding+of+multimeric+transmembrane+&#946;-barrel+proteins.">Assessing the outer membrane insertion and folding of multimeric transmembrane &#946;-barrel proteins.</a> Methods in Molecular Biology. , 157-167 (2015).</li><li>Mikula, K. M., Leo, J. C., &#321;yskowski, A., Kedracka-Krok, S., Pirog, A., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocation+domain+in+trimeric+autotransporter+adhesins+is+necessary+and+sufficient+for+trimerization+and+autotransportation.">The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation.</a> Journal of Bacteriology. 194 (4), 827-838 (2012).</li><li>Thomason, L. C., Costantino, N., Court, D. L., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+genome+manipulation+by+P1+transduction.">E. coli genome manipulation by P1 transduction.</a> Current Protocols in Molecular Biology. , (2007).</li><li>Franklin, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+in+gal+U+gene+of+E.+coli+blocks+phage+P1+infection.">Mutation in gal U gene of E. coli blocks phage P1 infection.</a> Virology. 38 (1), 189-191 (1969).</li><li>Jeong, H., 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=Genome+sequences+of+Escherichia+coli+B+strains+REL606+and+BL21(DE3).">Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).</a> Journal of Molecular Biology. 394 (4), 644-652 (2009).</li><li>Greene, E. C. <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+Alignment+during+Homologous+Recombination.">DNA Sequence Alignment during Homologous Recombination.</a> Journal of Biological Chemistry. 291 (22), 11572-11580 (2016).</li><li>Watt, V. M., Ingles, C. J., Urdea, M. S., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homology+requirements+for+recombination+in+Escherichia+coli.">Homology requirements for recombination in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (14), 4768-4772 (1985).</li><li>Ho, T. D., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterohemorrhagic+Escherichia+coli+O157:H7+gal+mutants+are+sensitive+to+bacteriophage+P1+and+defective+in+intestinal+colonization.">Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization.</a> Infection and Immunity. 75 (4), 1661-1666 (2006).</li><li>Ikeda, H., Tomizawa, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducing+fragments+in+generalized+transduction+by+phage+P1.+I.+Molecular+origin+of+the+fragments.">Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments.</a> Journal of Molecular Biology. 14 (1), 85-109 (1965).</li><li>Murooka, Y., Harada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+host+range+of+coliphage+P1+and+gene+transfer+from+enteric+bacteria+to+other+Gam-negative+bacteria.">Expansion of the host range of coliphage P1 and gene transfer from enteric bacteria to other Gam-negative bacteria.</a> Applied and Environmental Microbiology. 38 (4), 754-757 (1979).</li><li>Goldberg, R. B., Bender, R. A., Streicher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+selection+for+P1-sensitive+mutants+of+enteric+bacteria.">Direct selection for P1-sensitive mutants of enteric bacteria.</a> Journal of Bacteriology. 118 (3), 810-814 (1974).</li><li>O'Connor, K. A., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coliphage+P1-mediated+transduction+of+cloned+DNA+from+Escherichia+coli+to+Myxococcus+xanthus:+use+for+complementation+and+recombinational+analyses.">Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses.</a> Journal of Bacteriology. 155 (1), 317-329 (1983).</li></ol>

Los autores no tienen nada que revelar.

Transducción de P1 es un método rápido, robusto y fiable para la generación de canceladuras del gene de e. coli. Esto se demuestra aquí por transducción de un mutante de eliminación de tamA de una cepa de Keio donante a un beneficiario derivado BL21. Las principales etapas en el proceso de transducción de señales son la producción de la transducción de lisado, la transducción de sí mismo, la supresión de los cassettes de resistencia de Kan y la verificación del knock-out por PCR. En total...

Una primera aproximación común para estudiar la función de un gen es realizar mutagénesis de knock-out y observar el fenotipo resultante. Esto también se llama genética reversa. La bacteria e. coli ha sido el caballo de batalla de la biología molecular durante los últimos años 70 o así, debido a la facilidad de su cultivo y su receptividad a la manipulación genética1. Varios métodos han sido desarrollados para producir deleciones del gen en e. coli, incluyendo marcador cambio mutagénesis2,3 y, más recientemente, recombineering usando la λ rojo o ET Rac sistemas4,5 , 6.
En un sistema ampliamente utilizado, secuencias de codificación de genes individuales se sustituyen por una cinta de resistencia a los antibióticos que más adelante puede ser suprimida del cromosoma5,7. Las secuencias de codificación son sustituidas, por ejemplo por una cinta de resistencia kanamicina (Kan), que es flanqueada por los sitios de destino (FRT) flippase (FLP) reconocimiento a ambos lados. Los sitios FRT son reconocidos por el recombinase FLP, que media específica recombinación entre los sitios FRT conduce a la supresión de la cassette de Kan. De esta manera, se logra un borrado completo de la secuencia de codificación de un gen determinado, dejando sólo una secuencia mínima cicatriz de aproximadamente 100 pares de bases (PB) (figura 1).
A poco más de una década atrás, se desarrolló la llamada colección de Keio. Se trata de una biblioteca bacteriana basada en un estándar de laboratorio cepa K12 de e. coli , donde casi todos los genes no esenciales fueron borrados individualmente por λ recombinación rojo7,8. Los clones dentro de cada colección tienen una secuencia de codificación substituida con una cinta de resistencia los Kan. La colección de Keio ha demostrado para ser una herramienta útil para muchas aplicaciones9. Un tal uso es la producción de mutantes de deleción en otras cepas de e. coli . El cassette de Kan de un clon de eliminación dada puede ser movilizado por transducción generalmente de bacteriófagos, como P110,11,12,13,14. Un stock de fagos preparado a partir de tal tensión puede utilizarse para infectar a un recipiente e. coli cepa de interés, donde en una frecuencia baja pero confiable el Kan región que contiene el cassette puede ser incorporado en el genoma receptor por recombinación homóloga (Figura 2). Transductants pueden ser seleccionados para el crecimiento en el medio que contiene Kan. Después de esto, si se desea el retiro de la cinta de resistencia a los antibióticos, el recombinase FLP se puede suministrar a la cepa transductant en trans. Después de curar el plásmido que contiene el FLP, que lleva un marcador de resistencia a ampicilina (Amp), clones de Kan y sensible a Amp son evaluados para, y la supresión correcta de la secuencia de codificación de tipo salvaje y el cassette de Kan son verificados por PCR de Colonia.
Aquí, se presenta un protocolo detallado, describiendo cada uno de los pasos en la producción de un knock-out cepa de e. coli , basado en la estrategia que se ha señalado anteriormente. Por ejemplo, se demostró una canceladura del gene del tamA . tamA codifica una proteína de β-barril de membrana externa que es una parte del transporte y montaje módulo (TAM), que participa en la biogénesis de ciertas proteínas autotransporter y pili15,16,17. Esta cepa knock-out entonces fue utilizada para examinar el efecto de la supresión de tamA en la biogénesis de dos autotransporter trímero adhesinas (TAAs), la adhesina de Yersinia YadA y la inmunoglobulina (Ig) de la e. coli -vinculante EibD TAA 18,19.

<table><tbody><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BW25113</td><td>NIG</td><td>ME6092</td><td>Wild-type strain of Keio collection</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Merck</td><td>69450-3</td><td>Expression strain</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21DABCF</td><td>Addgene</td><td>102270</td><td>Derived from BL21(DE3)</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; JW4179</td><td>NIG</td><td>JW4179-KC</td><td>&lt;em&gt;tamA&lt;/em&gt; deletion mutant</td></tr><tr><td>P1 &lt;em&gt;vir&lt;/em&gt;</td><td>NIG</td><td>HR16</td><td>Generally transducing bacteriophage</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pCP20</td><td>CGSC</td><td>14177</td><td>conditionally replicating plasmid with FLP</td></tr><tr><td>pASK-IBA2</td><td>IBA GmbH</td><td>2-1301-000</td><td>expression vector</td></tr><tr><td>pEibD10</td><td>N/A</td><td>N/A</td><td>for production of EibD; plasmid available on request</td></tr><tr><td>pET22b+</td><td>Merck</td><td>69744-3</td><td>expression vector</td></tr><tr><td>pIBA2-YadA</td><td>N/A</td><td>N/A</td><td>for production of YadA; plasmid available on request</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid</td><td>ThermoFisher</td><td>33209</td><td></td></tr><tr><td>Agar</td><td>BD Bacto</td><td>214010</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Ampicillin</td><td>Applichem</td><td>A0839</td><td></td></tr><tr><td>Anhydrotetracycline</td><td>Abcam</td><td>ab145350</td><td></td></tr><tr><td>anti-collagen type I antibody COL-1</td><td>Sigma</td><td>C2456</td><td></td></tr><tr><td>Bovine collagen type I</td><td>Sigma</td><td>C9791</td><td></td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102382</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>102445</td><td></td></tr><tr><td>Di-sodium hydrogen phosphate</td><td>VWR</td><td>28029</td><td></td></tr><tr><td>DNA dye</td><td>Thermo</td><td>S33102</td><td></td></tr><tr><td>DNA molecular size marker</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>dNTP mix</td><td>New England Biolabs</td><td>N0447</td><td></td></tr><tr><td>ECL HRP substrate</td><td>Advansta</td><td>K-12045</td><td></td></tr><tr><td>EDTA</td><td>Applichem</td><td>A2937</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>24388</td><td></td></tr><tr><td>goat anti-mouse IgG-HRP</td><td>Santa Cruz</td><td>sc-2005</td><td></td></tr><tr><td>goat anti-rabbit IgG-HRP</td><td>Agrisera</td><td>AS10668</td><td></td></tr><tr><td>HEPES</td><td>VWR</td><td>30487</td><td></td></tr><tr><td>Isopropyl thiogalactoside</td><td>VWR</td><td>43714</td><td></td></tr><tr><td>Kanamycin</td><td>Applichem</td><td>A1493</td><td></td></tr><tr><td>Lysozyme</td><td>Applichem</td><td>A4972</td><td></td></tr><tr><td>Magnesium chloride</td><td>VWR</td><td>25108</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma</td><td>221279</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-lauroyl sarcosine</td><td>Sigma</td><td>L9150</td><td></td></tr><tr><td>Skim milk powder</td><td>Sigma</td><td>70166</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>27808</td><td></td></tr><tr><td>&lt;em&gt;tamA&lt;/em&gt; forward primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-GAAAAAAGG&lt;br /&gt;ATATTCAGGAGAAAATGTG-3&#039;</td></tr><tr><td>&lt;em&gt;tamA&lt;/em&gt; reverse primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-TCATAATT&lt;br /&gt;CTGGCCCCAGACC-3&#039;</td></tr><tr><td>Taq DNA polymerase</td><td>New England Biolabs</td><td>M0267</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Merck</td><td>106448</td><td></td></tr><tr><td>Tryptone</td><td>VWR</td><td>84610</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Yeast extract</td><td>Merck</td><td>103753</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose gel electrophoresis chamber</td><td>Hoefer</td><td>SUB13</td><td></td></tr><tr><td>Bead beater</td><td>Thermo</td><td>FP120A-115</td><td></td></tr><tr><td>CCD camera</td><td>Kodak</td><td>4000R</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Bio-Rad</td><td>165-2089</td><td></td></tr><tr><td>Electroporation unit</td><td>Bio-Rad</td><td>1652100</td><td></td></tr><tr><td>Gel imager</td><td>Nippon Genetics</td><td>GP-03LED</td><td></td></tr><tr><td>Incubating shaker</td><td>Infors HT</td><td>Minitron</td><td></td></tr><tr><td>Incubator</td><td>VWR</td><td>390-0482</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>Microwave oven</td><td>Samsung</td><td>CM1099A</td><td></td></tr><tr><td>PCR machine</td><td>Biometra</td><td>Tpersonal</td><td></td></tr><tr><td>PCR strips</td><td>Axygen</td><td>PCR-0208-CP-C</td><td></td></tr><tr><td>pH meter</td><td>Hanna Instruments</td><td>HI2211-01</td><td></td></tr><tr><td>PVDF membrane</td><td>ThermoFisher</td><td>88518</td><td></td></tr><tr><td>SDS-PAG electrophoresis chamber</td><td>ThermoFisher</td><td>A25977</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Beckman Coulter</td><td>B06322</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>SI-0236</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>D3006</td><td></td></tr><tr><td>Wet transfer unit</td><td>Hoefer</td><td>TE22</td><td></td></tr></tbody></table>

producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes

Una primera aproximación al estudio de la función de un gen desconocido en bacterias es crear un knock-out de este gen. Aquí, describimos un protocolo robusto y rápido para la transferencia de mutaciones de la canceladura de genes de una cepa de Escherichia coli a otra mediante la transducción generalizada con el bacteriófago P1. Este método requiere que la mutación sea seleccionable (p. ej., basado en interrupciones del gen mediante inserciones de cassette antibiótico). Tales cintas antibióticas pueden movilizar de una cepa donante e introducen en una cepa receptora de interés rápidamente y generan fácilmente un mutante de canceladura del gene. El cassette de antibiótico puede diseñarse incluir flippase sitios de reconocimiento que permiten la supresión de la cinta por un recombinase específico para producir un knock-out limpiado con sólo una secuencia de ~ 100-base-par-larga cicatriz en el genoma. Demostrar el Protocolo por la anulación del factor conjunto implicado en la biogénesis autotransporter la codificación del gene de tamA y probar el efecto de esta eliminatoria en la biogénesis y la función de dos autotransporter trímero adhesinas. Aunque la canceladura del gene por transducción P1 tiene sus limitaciones, la facilidad y rapidez de su aplicación hacen una alternativa atractiva a otros métodos de canceladura del gene.

Generación de un tamA Knock-out de BL21ΔABCF:
La estrategia mencionada anteriormente se ha utilizado para producir una cepa derivada de BL21(DE3), una cepa de laboratorio estándar utilizada para la producción de la proteína, que es optimizada para producción de proteínas de membrana externa y llamada BL21ΔABCF21. Esta cepa carece de cuatro genes que codifican para proteínas de membrana exte...

Watch this Scientific Journal Video about Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes at JoVE.com

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producción de canceladuras del Gene en Escherichia coli por transducción de P1 con casetes de resistencia a los antibióticos

Aquí presentamos un protocolo para el uso de preexistentes antibiótico resistencia-cassette borrado construcciones como base para la fabricación de mutantes de deleción en otras cepas de e. coli . Tales mutaciones de la canceladura pueden ser movilizados e inserta en el correspondiente locus de una cepa receptora mediante transducción de bacteriófago P1.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

producing-gene-deletions-escherichia-coli-p1-transduction-with

Research

JoVE Journal

Genetics

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

17.0K vistas.  University of Oslo. Aquí presentamos un protocolo para el uso de preexistentes antibiótico resistencia-cassette borrado construcciones como base para la fabricación de mutantes de deleción en otras cepas de e. coli . Tales mutaciones de la canceladura pueden ser movilizados e inserta en el correspondiente locus de una cepa receptora mediante transducción de bacteriófago P1.

Video: Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Sitio específico de Ingeniería cromosoma bacteriano: ΦC31 integrasa mediada Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Investigación

Bioingeniería

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Prueba el papel de Multicopy plásmidos en la evolución de resistencia a los antibióticos

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Genética

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Cuantificación de la resistencia a los antibióticos mediada por plásmido en un enfoque de evolución experimental

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Creación de un denso Transposon inserción biblioteca usando Conjugación bacteriana en cepas enterobacterianas tales como Escherichia Coli o Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Inmunología e Infección

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeo bacterianas redes funcionales y vías en<em&gt; Escherichia Coli</em&gt; Utilizando matrices sintéticas genéticos

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Biología

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genoma escala supresiones de genes en<em&gt; Streptococcus sanguinis</em&gt; Por PCR de alto rendimiento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Generación de mutantes de eliminación de genes en el marco en Pseudomonas aeruginosa y pruebas para la atenuación de la virulencia en un modelo simple de infección de ratón

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Eliminación de genes sin marcadores por Floxed Cassette Allelic Exchange Mutagenesis en Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Generación de<em&gt; Enterobacter</em&gt; Sp. YSU auxótrofos Usando transposón mutagénesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Producción de canceladuras del Gene en Escherichia coli por transducción de P1 con casetes de resistencia a los antibióticos

Resumen

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Capítulos en este video

Sitio específico de Ingeniería cromosoma bacteriano: ΦC31 integrasa mediada Cassette Exchange (IMCE)

Prueba el papel de Multicopy plásmidos en la evolución de resistencia a los antibióticos

Cuantificación de la resistencia a los antibióticos mediada por plásmido en un enfoque de evolución experimental

Creación de un denso Transposon inserción biblioteca usando Conjugación bacteriana en cepas enterobacterianas tales como Escherichia Coli o Shigella flexneri

Mapeo bacterianas redes funcionales y vías en

Genoma escala supresiones de genes en

Generación de mutantes de eliminación de genes en el marco en Pseudomonas aeruginosa y pruebas para la atenuación de la virulencia en un modelo simple de infección de ratón

Eliminación de genes sin marcadores por Floxed Cassette Allelic Exchange Mutagenesis en Chlamydia trachomatis

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Fagos mediada por la entrega de apuntado sRNA Construye para derribar la expresión génica en