Agradecemos al Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, EE.UU.) para el envío de cepas bacterianas, plásmidos y protocolos necesarios. Agradecemos al Dr. John Glover (Departamento de Bioquímica de la Universidad de Toronto) para el uso del lector de placas SpecraMax. Reino Unido es el destinatario de un Nacional de Ciencias e Ingeniería de Investigación de Canadá (NSERC) Becas de Postgrado, de los Institutos Canadienses de Investigación en Salud del Programa Estratégico de Formación en la biología estructural de proteínas de membrana ligados a la enfermedad, y de la Universidad de Toronto comunión abierta. Este trabajo fue apoyado por una beca de los Institutos Canadienses de Investigación en Salud (MOP-67210) a WAH.

1) Reactivos y Equipos <ol><li> En primer lugar, preparar las tres soluciones siguientes: 1 ml de solución A, que se compone de 8 mM de L-lisina, 100 mM de sodio 2 - (N-morfolino) el ácido etanosulfónico (MES) pH 6,5, 0,2 mM de nucleótidos, que es el nucleótido o bien: difosfato de guanosina (GDP), guanosina trifosfato (GTP), guanosina 5&#39;-difosfato, 3&#39;-difosfato (ppGpp), o guanosina 5&#39;-trifosfato, 3&#39;-difosfato (pppGpp), 0,1 mM piridoxal 5-fosfato (PLP), y 1 mM β-mercaptoetanol / (β-ME). 1 ml de solución B que consiste en 100 mM de sodio pH 6,5 MES, 0,1 mM PLP, 1 mM β-ME, y 50 nM LdcI. LdcI fue purificado como se describe en Snider et al. 6 y Kanjee et al. 14. 1 ml de solución C, que es idéntica a la solución B, pero no contiene LdcI. </li><li> Preparar 100 ml de la solución de paro que consiste en 1 M de carbonato de sodio (10,6 g/100 ml) y la parte alícuota de 50 l en cada pocillo de una placa de poliestireno de 96 pocillos utilizando una pipeta multicanal. Añadir 30 l de agua a la placa y la cubierta entonces. Tenga en cuenta que la suma del volumen de agua añadida y el volumen de muestra extraída durante la reacción de la enzima debe ser igual a 50 mL. En este caso, 20 l de muestra de enzima reacción será eliminado. </li><li> Preparar 5 ml de solución TNBS a 10 mM mediante la dilución de 294 l de 5% (w / v) acciones TNBS con 4.706 l de agua, envuelva en papel aluminio y mantener en hielo. </li><li> Equilibre el termostato Eppendorf Plus en 37 ° C. Además el termostato tiene 24 pozos en 6 columnas. Ver Figura 2 para el esquema. Etiqueta de 1,5 ml tubos Eppendorf indicando la identidad de los nucleótidos que se pondrá a prueba (una por columna): GTP (columna A), el PIB (columna B), ppGpp (columna C), pppGpp (columna D), no nucleótidos (columna E ), y las muestras de proteínas (columna F). </li><li> Bastidor de varias cajas de 200 tips l tal que cada fila alterna se omite un total de cuatro hileras de puntas. Esto es necesario para el uso de la pipeta multicanal con el termostato Además heatblock durante el ensayo enzimático. </li><li> Equilibre Digital Heatblock (VWR) a 42 ° C. </li><li> Coloque una de 96 y 2,0 mL placa de polipropileno en el hielo para enfriar. </li></ol> 2) Ensayo LdcI <ol><li> Alícuota de 50 l de la solución A con el nucleótido correspondiente a 1,5 ml tubos Eppendorf en cada una de las cinco columnas de la ThermoStat Eppendorf. </li><li> Añadir 330 l de la solución B a tres tubos en la columna F y añadir 330 l de solución C (el control sin proteínas) en el tubo final en la columna F. </li><li> Equilibrar las soluciones a 37 ° C durante cinco minutos. Sugerencia: después de 5 minutos, cortar las tapas de los tubos en el centro de la calefacción de bloque para evitar que interfieran con la pipeta multicanal para ser utilizado junto. </li><li> El uso de un 50 a 50 l VWR multi-canal de la pipeta, transferir 50 L de los tubos en la columna F en cada uno de los tubos en las columnas AE. En marcha el cronómetro tan pronto como el primer tubo se mezclan. </li><li> A los 2, 4 y 6 minutos, retire 20 l de la muestra con la pipeta multicanal y añadir a la solución de paro. </li><li> Nota: las muestras de solución de parada puede ser cubierta en plástico y congelado a -20 ° C para su posterior procesamiento. Las placas se deben descongelar a temperatura ambiente antes de la reacción con TNBS. </li></ol> 3) TNBS reacción y desarrollo de color <ol><li> Añadir 50 l de 10 mM solución TNBS a la solución de dejar de utilizar la pipeta multicanal y luego se incuba a 42 ° C durante 6 minutos. La solución se volverá de color amarillo oscuro / naranja como el TNBS reacciona con la lisina y la cadaverina. </li><li> Después de 6 minutos, enfriar la placa de hielo para retrasar la reacción. </li><li> Sacar 100 l de la muestra utilizando un VWR 20-200 l multi-canal pipeta y con más de 2 ml de profundidad y placa que se enfrió en hielo. Transferencia de cubo de hielo a la fumehood. </li><li> Añadir 500 l de tolueno a cada pocillo utilizando una HandyStep (Marca) Pipeta de repetición y 12,5 ml punta de la pipeta (PLASTIBRAND). </li><li> Limpie cualquier exceso de tolueno con una Kimwipe y cubrir el plato con tiras de cinta de embalaje. Asegúrese de presionar firmemente hacia abajo para obtener un buen sello. Cubrir la placa de 96 pocillos con tapa plana y se agita vigorosamente durante 1 minuto y 30 segundos. </li><li> Deje las soluciones reposar unos 5 minutos. El TNP-cadaverina se encuentra ahora en la fase de tolueno superior, mientras que el TNP-lisina sigue siendo soluble en agua (ver Figura 1). </li><li> Eliminar 200 L de tolueno con el 20-200 l multi-canal de la pipeta en una placa de 96 pozos para la lectura. Nota: asegúrese de comprobar que cada muestra se está quitando es clara y no tiene ninguna de la fase acuosa inferior. Nota: consejos de uso de barreras para evitar daños a múltiples canales de pipeta por el tolueno. </li><li> Lea la absorbancia a 340 nm en un lector de placas SpectraMax 340. </li><li> Para limpiar la placa de cuarzo, enjuagar el tolueno con agua y coloque la placa de cuarzo en un granvidrio bandeja en la campana extractora de humos. Verter sobre 100 ml de una mezcla de 7:3 de 70% (v / v) de ácido nítrico: 95% (v / v) de etanol y la cubierta de la placa de cuarzo con una bandeja de vidrio segundos. Advertencia: la reacción es muy exotérmica protección contra el desgaste y explosivo a veces apropiada y asegurarse de que la banda campana extractora de humos está en la parte baja de 6 &quot;de nivel Después de enfriar, lavar el ácido nítrico con agua y etanol al 95%.. </li></ol> 4) Los resultados representativos 1) cadaverina curva estándar (Figura 3) El ensayo se realizó como se describe, pero sin ningún tipo de L-lisina o LdcI y las concentraciones en lugar de varios cadaverina se utilizaron para determinar empíricamente el rango lineal del ensayo. El ensayo fue lineal hasta una DO 340 de 0.25, que corresponde a ~ 22 nmoles de cadaverina (Figura 3). 2) Actividad de LdcI (Figura 4) La actividad de LdcI solo se determinó que era 153,5 (± 18,1) nmoles cadaverina min-1 mg LdcI-1 a pH 6,5. La actividad de LdcI no se ve afectada en presencia de 100 mM GTP o el PIB, pero está fuertemente inhibida (&gt; 10 veces) en presencia de pppGpp y ppGpp (Figura 4). <img src="/files/ftp_upload/2094/2094fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de la reacción LdcI. La reacción de descarboxilación de la L-lisina para generar CO 2 y la cadaverina se muestra así como la posterior reacción con TNBS a pH alto para generar N, N&#39;-bistrinitrophenylcadaverine (TNP-cadaverina) y N, N &quot;-bistrinitrophenyllysine (TNP-lisina). En base a Phan et al.11 <img src="/files/ftp_upload/2094/2094fig2.jpg" alt="Figura 2" /> Figura 2. La configuración del termostato Eppendorf. El diseño de las muestras en el termostato de 24 y Eppendorf se muestra junto con la descripción de los pasos de la prueba (se indican en negrita). Las flechas indican la transferencia de soluciones de reacción de acuerdo con el protocolo. La eliminación de la solución de reacción a la solución de parada en la placa de 96 pozos también se indica. <img src="/files/ftp_upload/2094/2094fig3.jpg" alt="Figura 3" /> Figura 3. Cadaverina curva estándar. La gráfica de la absorbancia a 340 nm frente a nmoles de cadaverina se muestra. Las barras de error representan la desviación estándar de al menos tres mediciones independientes. La línea de mejor ajuste también se muestra y tiene una R2 de 0,996. <img src="/files/ftp_upload/2094/2094fig4.jpg" alt="Figura 4" /> Figura 4. LdcI resultados del ensayo. Un gráfico de la tasa de actividad LdcI (en nmoles cadaverina producido min-1 mg-1 LdcI) se muestra en la presencia de GTP M 100, el PIB, pppGpp, ppGpp y en ausencia de nucleótidos. Los nucleótidos respuesta estrictas (p) ppGpp son capaces de inhibir la actividad LdcI significativamente. Las barras de error representan la desviación estándar de al menos seis mediciones independientes.

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	<li>Gale, E. F., Epps, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+pH+of+the+medium+during+growth+on+the+enzymic+activities+of+bacteria+(Escherichia+coli+and+Micrococcus+lysodeikticus)+and+the+biological+significance+of+the+changes+produced.">The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced.</a> Biochem J. 36, 600-618 (1942).</li><li>Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+acid+resistance:+tales+of+an+amateur+acidophile.">Escherichia coli acid resistance: tales of an amateur acidophile.</a> Nat Rev Microbiol. 2, 898-907 (2004).</li><li>Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+acid+resistance+in+Escherichia+coli.">Control of acid resistance in Escherichia coli.</a> J Bacteriol. 181, 3525-3535 (1999).</li><li>Iyer, R., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-agmatine+antiporter+in+extreme+acid+resistance+in+Escherichia+coli.">Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.</a> J Bacteriol. 185, 6556-6561 (2003).</li><li>Sabo, D. L., Boeker, E. A., Byers, B., Waron, H., Fischer, E. 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En el ensayo de lisina descarboxilasa, TNBS se hace reaccionar con las aminas primarias de L-lisina y la cadaverina para formar TNP-lisina y TNP-cadaverina aductos (Figura 1). Debido a la presencia del grupo ácido carboxílico de TNP-lisina, este aducto permanece soluble en agua, mientras que el TNP-cadaverina, que carecen del grupo ácido carboxílico, es capaz de partición en tolueno 11. Este tipo de ensayo puede ser utilizado más ampliamente en otros tipos de aminoácidos que la pérdida de un grupo ác...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2,4,6-trinitrobenzensulfonic acid </td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2297</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mM pyridoxal 5'-phosphate (PLP)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P9255</td>
<td>&nbsp;</td>
<tr>
<td>Cadaverine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>D22606</td>
<td>&nbsp;</td>
<tr>
<td>96 well polystyrene plates</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96-well quartz plate</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>VWR Digital Heatblock</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>ThermoStat Plus</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2.0 mL 96-well polypropylene plates</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>P-DW-20-C</td>
<td>&nbsp;</td>
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<td>Handy Step Repeat Pipettor</td>
<td>&nbsp;</td>
<td>Brand</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>12.5 mL Repeat Pipettor Tips</td>
<td>&nbsp;</td>
<td>Brand (Plastibrand)</td>
<td>702378</td>
<td>&nbsp;</td>
<tr>
<td>SpectraMax 340PC Plate Reader</td>
<td>&nbsp;</td>
<td>SpectraMax</td>
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an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

Escherichia coli es una bacteria entérica que es capaz de crecer en un amplio rango de valores de pH (pH 5-9) 1 e, increíblemente, es capaz de sobrevivir ácido extremas tensiones incluyendo el paso por el estómago de mamíferos en donde el pH puede caer tan bajo como pH 1 - 2 2. Para permitir una gama tan amplia de la supervivencia de pH ácido, E. coli posee cuatro descarboxilasas diferentes inducible descarboxilación de aminoácidos que los ácidos amino sustrato en forma de protones dependiente de lo que aumenta el pH interno. El descarboxilasas incluyen el ácido glutámico descarboxilasas Gada y GadB 3, la arginina descarboxilasa Adia 4, la lisina descarboxilasa LdcI 5, 6 y la ornitina decarboxilasa SPEF 7. Todas estas enzimas utilizan piridoxal-5&#39;-fosfato como un co-factor 8 y funcionan en conjunto con la membrana interna de productos sustrato antiporters que eliminar los productos de descarboxilación al medio externo a cambio de sustrato fresco 2. En el caso de LdcI, el antiporter lisina cadaverina se llama Cadb. Recientemente, se determinó la estructura cristalina de rayos X de LdcI a 2,0 Å, y descubrimos una novela pequeña molécula unida a la guanosina LdcI respuesta regulador estricto 5&#39;-difosfato, 3&#39;-difosfato (ppGpp) 14. La respuesta estrictas ocurre cuando las células en crecimiento exponencial experiencia de la privación de nutrientes o una de una serie de otros factores de estrés 9. Como resultado, las células producen ppGpp que conduce a una cascada de señalización que culmina en el cambio de un crecimiento exponencial a fase estacionaria del crecimiento 10. Hemos demostrado que ppGpp es un inhibidor específico de LdcI 14. A continuación se describe el ensayo de lisina descarboxilasa, una modificación del ensayo desarrollado por Phan et al. 11, que hemos utilizado para determinar la actividad de LdcI y el efecto de pppGpp / ppGpp en esa actividad. La reacción de descarboxilación LdcI elimina el grupo α-carboxilo de la L-lisina y produce dióxido de carbono y la cadaverina poliamina (1,5-diaminopentano) 5. L-lisina y la cadaverina puede reaccionar con el ácido 2,4,6-trinitrobenzensulfonic (TNBS) a un pH elevado para generar N, N&#39;-bistrinitrophenylcadaverine (TNP-cadaverina) y N, N-bistrinitrophenyllysine (TNP-lisina), respectivamente 11. El TNP-cadaverina se puede separar de la TNP-lisina como el primero es soluble en disolventes orgánicos como el tolueno y el segundo no lo es (ver Figura 1). El rango lineal del ensayo fue determinado empíricamente por medio purificada cadaverina.

Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

La actividad de la descarboxilasa inducible lisina es controlada por la reacción del sustrato L-lisina y el producto cadaverina con 2,4,6-trinitrobenzensulfonic ácido para formar aductos que tienen una solubilidad diferencial en tolueno.

Un ensayo para medir la actividad de Escherichia coli Inducible lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

an-assay-for-measuring-activity-escherichia-coli-inducible-lysine

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

14.1K vistas.  University of Toronto. La actividad de la descarboxilasa inducible lisina es controlada por la reacción del sustrato L-lisina y el producto cadaverina con 2,4,6-trinitrobenzensulfonic ácido para formar aductos que tienen una solubilidad diferencial en tolueno.

Video: An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

La medición de la resistencia a la flexión de las células bacterianas usando una trampa óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Investigación

Biología

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cribado de alto rendimiento de la actividad endoglucanasa por hongos en Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Aplicación de MassSQUIRM para mediciones cuantitativas de la actividad desmetilasa Lisina

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Protocolo rápido para la preparación de electrocompetentes<em&gt; Escherichia coli</em&gt; Y<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Los protocolos para la implementación de un<em&gt; Escherichia coli</emSistema&gt; Basado TX-TL-Cell de Libre Expresión de la biología sintética

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

La detección de vivo<em&gt; Escherichia coli</em&gt; O157: H7 células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Las multifacéticas beneficios de la proteína co-expresión en<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

La incorporación de residuos específicos de no canónicos aminoácidos en las proteínas modelo usando una<em&gt; Escherichia coli</em&gt; Sistema de transcripción-traducción libre de células

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medición de actividad enzimática de la lactasa en el laboratorio de enseñanza

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...