Este trabajo fue financiado por la Gordon and Betty Moore Foundation, y por la Iniciativa de UNCF / Merck Ciencia.

1. Detección preparación placa <ol><li> Agregar 25 g de crecimiento medio LB, agar 15 g, y 1,5 g de carboximetilcelulosa (CMC) del sustrato de 1 litro de agua destilada y autoclave para esterilizar. </li><li> Después del autoclave, deje a medio enfriar y añadir los antibióticos adecuados, y IPTG a una concentración final de 100μM. </li><li> Vierta 200 ml de medio cada uno en cinco grandes platos de Petri cuadrados / bandejas de bioensayo (240 x 240 x 20 mm o más). Permita que las placas se seque por completo. </li></ol> 2. Endoglucanasa generación de biblioteca y de la pantalla <ol><li> Generar una biblioteca de dominio endoglucanasa catalítico (s). Esto se puede lograr de muchas maneras (por ejemplo, dirigida a un sitio o mutagénesis aleatoria, la recombinación, etc) y se determina por el investigador. </li><li> Clon de la biblioteca en un vector de tal manera que están bajo el control del promotor T7 con el operador lac, y contienen una secuencia señal pelB para orientar a la periplasma. Un ejemplo de un vector adecuado es pET22b (+) de Novagen. </li><li> Transformar la biblioteca en endoglucanasa E. coli cepa BL21 (DE3) y la placa de colonias aisladas. </li><li> Incubar transformación durante la noche a 37 ° C. </li><li> Con mondadientes estéril, inocular los clones en placas de 96 pozos profundos, así que contenían medio LB 400μL complementado con los antibióticos adecuados. Se incuba a 37 ° C durante la noche con agitación a 250 rpm. </li><li> Añadir glicerol estéril a 96 culturas y noche a la mañana a una concentración final del 10% para el almacenamiento a largo plazo a -80 ° C. Este será el plato principal. </li><li> El uso de un sello de 96 pines, se replican cultivos en placa durante la noche sobre las placas de detección que contiene IPTG y CMC. Hasta 5 grupos de cultivos durante la noche puede ser estampado en cada plato de detección. </li><li> Etiqueta de las placas de selección de modo que la identidad y la orientación de cada placa de 96 pozos que se conoce. </li><li> Incubar las bibliotecas sellada a temperatura ambiente (17-25 ° C) durante la noche o hasta que las colonias visibles se han formado. </li><li> Enjuagar la placa con agua destilada hasta que todos los restos de las colonias se eliminan. </li><li> Vierta Congo Roja (CR, 0,5% en agua) en la placa de lavado. Asegúrese de añadir sólo lo suficiente CR para cubrir la placa. </li><li> Incubar 15 minutos a temperatura ambiente. No exceder los 15 minutos de incubación CR. Ya los tiempos de incubación dará lugar a halos menos clara. </li><li> Vierta CR y añadir una cantidad abundante (más que suficiente para cubrir la placa) de NaCl 1M. </li><li> Incubar 15 minutos a temperatura ambiente. </li><li> Vierta NaCl para revelar halos CMC degradación. Para una mejor resolución de los halos, ya los tiempos de incubación se lava con NaCl NaCl o múltiple puede ser requerida. </li><li> Con un palillo estéril, elegir clones enzimas activas de la placa principal para una mayor caracterización. </li></ol> 3. Los resultados representativos: Un ejemplo de la lectura de esta pantalla de alto rendimiento se muestra en la Figura 3. Los clones pueden ser identificados como inactivos, poco activo, y muy activa en función del tamaño de las zonas de degradación. Esto es especialmente útil si una enzima de actividad conocida se incluye en la biblioteca de referencia. Como se muestra aquí, la identificación de enzimas activas es robusta y reproducible. Sin embargo, tenga en cuenta que el etiquetado adecuado de las placas de detección es muy importante. El investigador debe ser capaz de identificar las variantes activas después de lavar las colonias de distancia, con el fin de elegir el plato principal se almacenan a -80 ° C para una mayor caracterización. Por último, es difícil evaluar a priori la relación entre las actividades en substratos artificiales vs natural. Por lo tanto, el investigador debe poner a prueba todos los clones positivos en el material relacionado con la industria para determinar la aptitud de proteínas. <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="Figura 1"/> Figura 1. Esquema de los del procedimiento de selección. Las células se transforman con una biblioteca endoglucanasa y chapada selectiva de colonias aisladas. Clones recogidos y crecido durante la noche en placas de 96 pozos profundos y para (1) de almacenamiento a -80 ° C y (2) placas réplica en placas con CMC y IPTG para inducir la expresión endoglucanasa. Clones activos son recogidos de la placa principal se almacenan a -80 ° C. <img src="/files/ftp_upload/2942/2942fig2.jpg" alt="Figura 2"/> Figura 2. Clonación y expresión de hongos dominios catalíticos en E. coli. (A) endoglucanasa vector de expresión. Los genes fueron clonados en pET22b (+) (Novagen) bajo el control del promotor T7. (B) del panel derecho: endoglucanasa expresan BL21 (DE3) colonias. Panel de la izquierda: CMC halos de degradación después del lavado y el desarrollo con Rojo Congo. <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="Figura 3"/> Figura 3. Resultados de las muestras de CMC / Congo pantalla roja. Las imágenes tomadas de placas de detección después del lavado y el desarrollo de Rojo Congo. Todas las colonias en screening placas eran de un tamaño similar.

<ol>
	<li>Atsumi, S., Hanai, T., Liao, 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=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, P. J. <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+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

No hay conflictos de interés declarado.

El protocolo aquí descrito permite la identificación rápida y el rendimiento elevado de enzimas activas, con una manipulación mínima. La detección de actividad es bastante sensible, y cualitativamente refleja la cantidad de actividad dentro de la célula. Su facilidad de uso hace que este método es adecuado para una amplia gama de bibliotecas de la enzima, sólo limitado por la expresión funcional en E. coli. Además, la detección de actividad de este tipo no se limita a las celulasas fúngicas, pero puede ser adaptado para cualquier actividad endoglucanasa, o cualquier otra actividad celulasa para el cual no es un sustrato soluble (como CMC).

<table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Número del producto </th></tr><tr><td> IPTG </td><td> Sigma </td><td> I1284 </td></tr><tr><td> Rojo Congo </td><td> Sigma </td><td> C6277 </td></tr><tr><td> Carboximetil celulosa </td><td> Sigma </td><td> 360384 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S1679 </td></tr><tr><td> Bacto-agar </td><td> BD Biosciences </td><td> 214030 </td></tr><tr><td> Placas de bioensayo </td><td> Thermo Scientific </td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

Celulasa enzimas (endoglucanasas, celobiohidrolasas, y β-glucosidasas) hidrolizar la celulosa en azúcares componentes, que a su vez se puede convertir en una alcoholes carburantes. El potencial para la hidrólisis enzimática de biomasa celulósica para suministrar energía renovable ha intensificado sus esfuerzos para diseñar celulasas para 2 económica de producción de combustible. De particular interés son las celulasas fúngicas 3.8, que ya están siendo utilizados en la industria de los alimentos y el procesamiento de textiles. La identificación de variantes de activos entre una biblioteca de celulasas mutante es fundamental para el proceso de ingeniería; mutantes activo puede realizar más pruebas para mejorar las propiedades y / o sometidas a mutagénesis adicionales. Ingeniería eficiente de las celulasas fúngicas se ha visto obstaculizada por la falta de herramientas genéticas para los organismos nativos y por las dificultades en la expresión de las enzimas en los ejércitos heteróloga. Recientemente, Morikawa y colaboradores desarrollaron un método para expresar en E. coli los dominios catalíticos de endoglucanasas de H. jecorina 3,9, un hongo industrial importante con la capacidad de secretar celulasas en grandes cantidades. Funcionales E. expresión coli también se ha informado de celulasas de otros hongos, como Macrophomina phaseolina 10 y Phanerochaete chrysosporium 11-12. Se presenta un método de cribado de alto rendimiento de la actividad endoglucanasa hongos en E. coli. (Fig. 1) Este método utiliza el común microbiana colorante rojo Congo (RC) para visualizar la degradación enzimática de la celulosa carboximetil (CMC) por las células que crecen en medio sólido. El ensayo de la actividad requiere de reactivos de bajo costo, mínima manipulación, y da resultados sin ambigüedades como las zonas de degradación (&quot;halos&quot;) en el sitio de la colonia. Aunque una medida cuantitativa de la actividad enzimática no se puede determinar por este método, hemos encontrado que el tamaño del halo de correlaciona con la actividad enzimática en el total de células. Además de la caracterización de cada uno de los clones positivos se determine, la aptitud relativa de proteínas. Tradicionales de bacterias de células enteras CMC / CR 13 ensayos de actividad implica verter CMC agar que contiene a las colonias, que está sujeta a la contaminación cruzada, o culturas incubando en los pozos de agar CMC, que es menos susceptible a la experimentación a gran escala. Aquí se presenta un protocolo mejorado, que modifica los actuales métodos de lavado de 14 para la actividad de celulasa: las células cultivadas en placas de agar CMC se retiran antes de la tinción CR. El protocolo reduce de forma significativa la contaminación cruzada y es altamente escalable, que permite la detección rápida de miles de clones. Además de la H. jecorina enzimas, que han expresado y se seleccionan variantes endoglucanasa de la aurantiacus Thermoascus y Penicillium decumbens (como se muestra en la Figura 2), lo que sugiere que este protocolo se aplica a las enzimas a partir de una amplia gama de organismos.

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Se describe un método de bajo costo, de alto rendimiento para la detección de hongos en la actividad endoglucanasa<em&gt; E. coli.</em&gt; El método se basa en una lectura simple y visual de la degradación del sustrato, no requiere purificación de la enzima, y ​​es altamente escalable. Esto permite la detección rápida de las grandes bibliotecas de variantes de la enzima.

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 ...

high-throughput-screening-fungal-endoglucanase-activity-escherichia

Nanyang Technological University

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.5K vistas.  California Institute of Technology. Se describe un método de bajo costo, de alto rendimiento para la detección de hongos en la actividad endoglucanasa E. coli. El método se basa en una lectura simple y visual de la degradación del sustrato, no requiere purificación de la enzima, y ​​es altamente escalable. Esto permite la detección rápida de las grandes bibliotecas de variantes de la enzima.

Video: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine 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 acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

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

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

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 ...

Investigación

Biología

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Selección de alto rendimiento y biosensores fluorescentes con C. elegans Las cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

De alto rendimiento para los moduladores de molécula pequeña de tráfico de perfeccionamiento activo Canales rectificador de potasio

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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 ...

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. ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expresión cuantitativa y Purificación Aplicada a Recombinante Disulfide ricos en proteínas del veneno Producido en<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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

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La detección de vivo

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Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados