Este trabajo fue financiado en parte por el Departamento de Energía de EE.UU. de los Grandes Lagos Centro de Investigación en Bioenergía (DOE Oficina de Ciencia BER DE-FC02-07ER64494) y conceder DE-FG02-91ER200021 del Departamento de Energía de EE.UU., Oficina de Ciencias Básicas de Energía, División de de Ciencias Químicas, Geociencias y Ciencias Biológicas. Agradecemos a John Scott-Craig y Borrusch Melissa por sus contribuciones materiales y conceptuales.

1. Producción de enzimas <ol><li> La producción de proteínas en Pichia pastoris mediante la clonación de los genes correspondientes en vectores pPICZ o pPICZα y su transformación en P. pastoris. Se cultivan las células en 300 ml en lotes desconcertado matraces de 500 ml con la inducción de metanol cada 24 horas (Banerjee et al., 2010a). </li><li> Concentrar y desalinizar los filtrados del cultivo por filtración de flujo tangencial. </li><li> Tienda de las enzimas en pequeñas alícuotas de 20% de glicerol a -80 ° C. El rango de concentración es 1.10 mg / ml. </li></ol> 2. Diseño del Experimento <ol><li> Introduzca el número de enzimas para la prueba, su superior e inferior de proporciones (por ejemplo, una menor proporción de 5% para las enzimas del núcleo), y el tipo de diseño experimental (por ejemplo, cuadrática o cúbica) en el diseño Expertsoftware. </li><li> Calcular la cantidad en mg de cada enzima que se añade a hacer una carga total de 15 mg / g glucano, y después calcular la cantidad de cada enzima en l que añadir acada reacción. </li><li> Generar hojas de cálculo Excel especificando material de laboratorio de origen, los pozos de la fuente, material de laboratorio de destino, los pozos de destino, y los volúmenes de transferencia para la distribución de agua y las enzimas de acuerdo con el diseño experimental y la importación en el software Biomek FXP utilizando la función de transferencia-de-archivo. </li></ol> 3. La hidrólisis enzimática <ol><li> Suspender la suspensión de la biomasa en 50 mM de citrato sódico, pH 4,8, que contiene 5 mg / ml de tetraciclina y 5 cicloheximida mg / ml, en el embalse de paddle. </li><li> Robot pipeta 200 ul de la mezcla en cada pocillo de una placas de 96 pocillos de reacción de pozo profundo con Span-8 1000-l puntas de pipeta con los últimos 0,5 cm cortado. Mezclar la pasta una vez a 100 l / seg y luego aspirar el mismo volumen de la reserva paddle y se distribuye en las placas. </li><li> Prescindir de las enzimas en el mismo plato con el programa entró en el FX Beckman. </li><li> Sellar las placas con capmats perforables. </li><li> Invertir y toquelas placas en varias ocasiones para superar la tensión superficial. Coloque las placas a 50 ° C durante 48 horas en la hibridación incubadora girando a 10 rpm. </li></ol> 4. Determinación de la Glc y XYL <ol><li> Centrifugar las placas de reacción durante 3 minutos a 1250 xg en una centrífuga de basculante. </li><li> Transferencia de 100 l del sobrenadante en regulares placas de 96 pocillos con la vaina de la AP96 FX Biomek. </li><li> Las placas se incuban a 90-95 ° C durante 10 minutos para inactivar las enzimas, y luego se centrifuga a 1250 xg durante 30 segundos. </li><li> Para las mediciones de Glc, la transferencia de 12 l de los sobrenadantes en regulares placas de 96 pocillos. Añadir 192 l de la glucosa oxidasa / peroxidasa (goPod) reactivo. Las placas se incuban a 50 ° C durante 20 minutos y leer las absorbancias a 510 nm en un lector de microplacas. En blanco se mezcla de reacción sin enzima. </li><li> Para las mediciones de XYL, la transferencia de 4 l en placas de 384 pocillos. Añadir los reactivos de ensayo y leer las placas a 340 nm. En blancoes la mezcla de reacción sin enzima. </li></ol> 5. Análisis de Datos <ol><li> Convertir los valores de absorbancia a su Glc% y los rendimientos XYL basada en el conocido Glc y contenido XYL de la biomasa original. Importación de estos porcentajes, las respuestas en el software Design-Expert. Compruebe la paramenters estadísticos para asegurarse de que los criterios para un modelo sólido que se cumplan. </li><li> Confirman la predicción mejor modelo experimental. </li></ol> 6. Los resultados representativos con GENPLAT: (1) Creación de mezclas optimizadas de cada uno de proteínas puras. Los resultados indican que las enzimas son importantes, y en qué proporciones, y también que las enzimas no son importantes. Algunas proteínas que son abundantes en la T. reesei secretoma (Nagendran et al., 2009), como Cip1 y CIP2, parecen desempeñar ningún papel en la liberación del maíz Glc pretratados por la expansión de fibra amoniaco (AFEX) o alcalino hyd rastrojo<html> Rogen peróxido (AHP) (Banerjee et al., 2010b). Por otro lado, algunas proteínas son inesperadamente importante. Por ejemplo, endo-xilanasas de las familias de glicosil hidrolasa 10 y 11 son importantes para la liberación Glc, una xilanasa no puede sustituir a la otra (Banerjee et al, 2010b, c).. Cel61A, una proteína de menor importancia en la T. reesei secretoma, es muy importante para Glc pero no XYL liberación. Algunas enzimas son importantes para la liberación de ambos Glc y XYL, mientras que otros sólo son necesarios para uno u otro (Banerjee et al., 2010c). Una mezcla sintética que contiene 16 componentes, cada uno a una concentración de 0,94 mg / ml (es decir, una mezcla no optimizado), lanzado el 38% de la disponible Glc de AFEX pretratados DDG. En condiciones de hidrólisis idénticas, una mezcla optimizada, en la que las concentraciones de los 16 componentes del 0% al 32%, dado a conocer el 52% de la Glc (Fig. 2). Este experimento ilustra la utilidad de la construcción de mezclas definidas. _content &quot;&gt; (2) Creación de un cóctel optimizado para el pretratamiento diferentes / combinaciones de sustrato. actuales preparaciones de celulasa comerciales son de&quot; talla única para todos &quot;. En realidad, el mejor cóctel depende del sustrato y el pretratamiento. Un solo producto como Accellerase 1000 ofrece diferentes rendimientos de los diferentes sustratos tratados previamente de la misma manera, y desde el mismo sustrato expuestos a diferentes tratamientos previos (Fig. 1). Hemos utilizado GENPLAT para optimizar las mezclas de enzimas puras para múltiples tratamientos previos y materias primas mutiple (Banerjee et al., 2010c). Figura 2 muestra cómo la proporción óptima de la enzima de 16 enzimas puras es diferente para el rastrojo de maíz AFEX pretratados, AHP pretratados rastrojo de maíz y DDG AFEX pretratados. Sólo el 11 por enzimas se muestra en la Fig. 2. porque el óptimo proporciones de los otros cinco (Cel61B, AbfB, Cip1, CIP2 y Cel12A) resultaron ser del 0% (Banerjee et al., 2010c). (3) Nuevos descubrimiento de enzimas </strong&gt;. La construcción de mezclas optimizadas de proteínas puras es un proceso continuo. Como las proteínas se dispone de nuevas en su forma pura (a partir de fuentes comerciales, por la expresión heteróloga, o por la purificación), pueden ser sometidas a ensayos mediante su inclusión en el conjunto optimizado actual. Nuevas proteínas que contribuyen a la liberación Glc o XYL pasan a formar parte de una nueva y optimizada. De esta manera, GENPLAT convierte en una plataforma para la identificación de nuevas enzimas para la deconstrucción de la biomasa. Hemos hecho los extractos de otros hongos que T. reesei y A. niger cultivado en diferentes sustratos. Un extracto especial impulsado Glc rendimiento cuando se añade a nuestra colección básica. La proteína responsable fue purificado y ha demostrado ser una hidrolasa novela glicosil no están presentes en cualquier preparado enzimático comercial (Banerjee y Walton, resultados no publicados). (4) El descubrimiento de mejores enzimas. Después de haber demostrado con GENPLAT que las enzimas son más importantes para la degradación de unbiomasa en particular, tenemos una mejor comprensión de las enzimas que debe ser el enfoque de mejora. Mejores ejemplos de las enzimas críticas se puede encontrar buscando en la naturaleza o fabricados por ingeniería de proteínas. (Una enzima podría ser &quot;mejor&quot; que su homólogo de T. reesei de varias maneras, dependiendo de la propiedad deseada, por ejemplo, actividad específica más alta, una mayor sinergia, disminución de la sensibilidad proteolítica, o la estabilidad térmica mejorada). GENPLAT ofrece una plataforma significativa para el ensayo de enzimas potencialmente mejor, ya que utiliza un cóctel complejo realista (importante porque no enzimática de biomasa funciona en forma aislada) y un sustrato realista (importante porque los resultados con pura celulosa u otros sustratos artificiales pueden no ser relevantes para lignocelulósicos naturales materiales). (5) la optimización de enzimas comerciales. Cantidades suficientes de enzimas puras no existen en el mundo real. Hasta que lo hagan, el etanol prolos productores deben confiar en los productos comerciales, tales como Accellerase 1000 y MultifectXylanase. Sin embargo, las mezclas de enzimas comerciales funcionan mejor que cualquier individuo, y GENPLAT se puede utilizar para el diseño optimizado de múltiples cócteles de enzimas comerciales. La Figura 3 muestra el uso de GENPLAT para optimizar una mezcla de cuatro preparados enzimáticos comerciales. Las proporciones óptimas para la liberación de Glc de AFEX pretratados DDG se encontró que el 47% Accellerase1000, el 27% Multifect pectinasa, el 22% Novozyme 188, y el 4% Multifect xilanasa (Banerjee et al., 2010c). Figura 3B muestra las diferencias en el rendimiento de AFEX Glc-DDG con cuatro preparados enzimáticos, la mezcla comercial optimizado ofrece un mayor rendimiento de Glc Accellerase1000 solo, SpezymeCP solo, o una mezcla sintética de 16 componentes. Esta experiencia también indica que la mezcla sintética de 16 componentes que falta uno o más enzimas necesarias para la óptima liberación Glc, que están presentes en uno o más de las enzimas comerciales. GENPLAT puede sered para identificar estos componentes. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Figura 1"/> Figura 1. No solo la preparación enzimática comercial es óptimo para todos los sustratos y pretratamientos todos. Cinco sustratos y tratamientos previos tres se compararon en las mismas condiciones de la molienda (0,5 mm de tamaño de partícula), la carga de la enzima (15 mg / g glucano), y las condiciones de hidrólisis (48 h, 50 ° C). A. La digestión de los diferentes sustratos pretratados AFEX con Accellerase 1000. digestión B. de rastrojo de maíz pretratadas con tres métodos diferentes con Accellerase 1000 (ver Banerjee et al., 2010c). <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Figura 2"/> Figura 2. Proporciones óptimas de 11 enzimas para la liberación de Glc de AFEX pretratados con el rastrojo de maíz (AFEX-CS), alcalinas de peróxido de hidrógeno pretratados con el rastrojo de maíz (AHP-CS), o AFEX pretratados secos destilargranos de res &quot;(AFEX-DDG). Los datos proceden de Banerjee et al. (2010c). Números en la parte superior de las barras son los rendimientos Glc como un porcentaje del total de Glc en la muestra de la biomasa. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Figura 3"/> Figura 3. A. El uso de GENPLAT para producir una mezcla optimizada de los cuatro preparados enzimáticos comerciales para la liberación de Glc de AFEX pretratados DDG. Multifect xilanasa hecho la contribución más pequeña (4%) y por lo tanto, se omitió en este diagrama ternario. Glc B. rendimientos de AFEX-DDG con tratamientos de enzimas diferentes en condiciones de hidrólisis idénticos (15 mg / g enzima glucano, 48 h, 50 ° C) . &quot;Cuatro componentes comerciales&quot; tiene las proporciones determinadas a partir del experimento se muestra en la fig. 3A.

<ol>
	<li>Anderson, M. J., Whitcomb, 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=.">.</a> DOE Simplified: Practical Tools for Effective Experimentation. , (2007).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+enzyme+mixtures+for+biomass+deconstruction:+production+and+optimization+of+a+core+set.">Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.</a> Biotechnol. Bioengineer. 106, 707-720 (2010).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Bongers, M., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+multi-component+enzyme+mixtures+for+deconstruction+of+lignocellulosic+biomass.">Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass.</a> Bioresour. Technol. 101, 9097-9105 (2010).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+optimization+of+enzyme+mixtures+for+deconstruction+of+diverse+pretreatment/biomass+feedstock+combinations.">Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.</a> Biotechnol. Biofuels. 3, 22-22 (2010).</li><li>Berlin, A., Maximenko, V., Gilkes, N., Saddler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+enzyme+complexes+for+lignocellulose+hydrolysis.">Optimization of enzyme complexes for lignocellulose hydrolysis.</a> Biotechnol. Bioeng. 97, 287-296 (2007).</li><li>Decker, S. R., Brunecky, R., Tucker, M. P., Himmel, M. E., Selig, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+screening+techniques+for+biomass+conversion.">High throughput screening techniques for biomass conversion.</a> Bioenerg. Res. 2, 179-192 (2009).</li><li>Gao, D., Chundawat, S. P., Krishnan, C., Balan, V., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixture+optimization+of+six+core+glycosyl+hydrolases+for+maximizing+saccharification+of+ammonia+fiber+expansion+(AFEX)+pretreated+corn+stover.">Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover.</a> Bioresour. Technol. 101, 2770-2781 (2010).</li><li>Harris, P. V., Welner, D., McFarland, K. C., Re, E., Poulsen, J. C. N., Brown, K., Salbo, R., Ding, H., Vlasenko, E., Merino, S., Xu, F., Cherry, J., Larsen, S. Y., LoLeggio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+lignocellulosic+biomass+hydrolysis+by+proteins+of+glycoside+hydrolase+family+61:+Structure+and+function+of+a+large,+enigmatic+family.">Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: Structure and function of a large, enigmatic family.</a> Biochemistry. 49, 3305-3316 (2010).</li><li>Kim, E., Irwin, D. C., Walker, L. P., 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=Factorial+optimization+of+a+six-cellulase+mixture.">Factorial optimization of a six-cellulase mixture.</a> Biotechnol. Bioeng. 58, 494-501 (1998).</li><li>King, B. C., Donnelly, M. K., Bergstrom, G. C., Walker, L. P., Gibson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+microplate+assay+system+for+quantitative+evaluation+of+plant+cell+wall-degrading+enzyme+activity+of+fungal+culture+extracts.">An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.</a> Biotechnol. Bioeng. 102, 1033-1044 (2009).</li><li>Nagendran, S., Hallen-Adams, H. E., Paper, J. M., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+genomic+potential+for+secreted+plant+cell-wall-degrading+enzymes+in+the+ectomycorrhizal+fungus+Amanita+bisporigera,+based+on+the+secretome+of+Trichoderma+reesei.">Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.</a> Fung. Genet. Biol. 46, 427-435 (2009).</li><li>Rosgaard, L., Pedersen, S., Langston, J., Akerhielm, D., Cherry, J. R., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+minimal+Trichoderma+reesei+cellulase+mixtures+on+differently+pretreated+barley+straw+substrates.">Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates.</a> Biotechnol. Prog. 23, 1270-1276 (2007).</li></ol>

No hay conflictos de interés declarado.

Es ampliamente reconocido que la reducción del costo de las enzimas es importante para el desarrollo de una industria de etanol lignocelulósico económica. Cócteles comerciales disponibles en la actualidad las enzimas son mezclas complejas y mal definido de muchas proteínas (Nagendran et al., 2009), y están adaptados principalmente para el uso de ácido pretratados con el rastrojo de maíz. Con el fin de acelerar el desarrollo de un cóctel de enzimas mejor, varios laboratorios han desarrollado plataformas de alto rendimiento para el descubrimiento y caracterización de enzimas. Los esfuerzos en esta área han incorporado uno o más de las siguientes propiedades también se encuentra en GENPLAT: robot de dispensación de las enzimas y lodos de la biomasa, el diseño estadístico de experimentos, y / o determinación automática de Glc y XYL (Berlin et al, 2007; Decker et. al, 2009;. Kim et al, 1998;. King et al, 2009).. GENPLAT extiende estos primeros esfuerzos, lo más importante en la complejidad de las mezclas de enzimas que pueden ser analizados desde un máximo de 6 componentes enlos estudios anteriores de más de 16 años en nuestros últimos trabajos (Banerjee et al., 2010c). Otras funciones clave de GENPLAT son el uso de una cámara de mezcla de bolas (depósito de remo) que puede mantener el rastrojo lodos suspendidos durante la distribución, mezcla suave durante la digestión de extremo a extremo de rotación, y la determinación colorimétrica automatizado de Glu y XYL.

Equipos y software que se utiliza: <ol><li> Design-Expert, versión 8.0 (Stat-Ease, Inc., Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP laboratorio de automatización de estaciones de trabajo (Beckman-Coulter, Fullerton, CA) </li><li> Biomek FXP software, la versión 3.3 (Beckman-Coulter, Fullerton, CA) </li><li> Paddle depósito (Modelo VP 756C-1P100, V &amp; P Scientific Inc., San Diego) </li><li> Placas, de 96 de profundidad (1,5 ml) y (ABgene, Fisher Scientific, Epson, Reino Unido) </li><li> Capmats perforable (EE.UU. Científico, Ocala, FL) </li><li> Rotación de hibridación incubadora (Modelo 5420, VWR) </li><li> Swinging-cubo de centrífuga (Modelo 5810R, Eppendorf, Hamburgo, Alemania) </li><li> Placas, Costar de 96 pocillos (ABgene, Fisher Scientific, Epson, Reino Unido) </li><li> Placas, 384-y (BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Reactivos utilizados: <ol><li> Pichia pastoris y vectores: cepa X33, y pPICZ y pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei y vectores: la tensión QM9414 (EE.UU. Departamento de Agricultura de Centro Nacional para la Investigación de Utilización Agrícola en Peoria, IL), y pCB1004 EV-(Banerjee et al, 2010b). </li><li> Accellerase 1000 (número de lote 1600844643) (Genencor, Inc., Rochester, NY) </li><li> Sodio 50 mM citrato, pH 4,8 </li><li> Tetraciclina stock: 10 mg / ml </li><li> Acciones cicloheximida: 10 mg / ml </li><li> GoPod kit de ensayo, el catálogo de K-gluc (Megazyme, Bray, Irlanda) </li><li> Xilosa kit de ensayo, el catálogo de K-xilosa (Megazyme, Bray, Irlanda) </li></ol>

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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Watch this Scientific Journal Video about GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization at JoVE.com

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT (Plataforma GLBRC la enzima) es una plataforma automatizada para el descubrimiento y optimización de los cócteles de enzimas para la degradación de la biomasa. Se puede adaptar a las materias primas y varias mezclas de enzimas que contienen múltiples componentes.

GENPLAT: una plataforma automatizada para el descubrimiento de enzimas de la biomasa y la optimización Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Research

JoVE Journal

Bioengineering

15.3K vistas.  Michigan State University. GENPLAT (Plataforma GLBRC la enzima) es una plataforma automatizada para el descubrimiento y optimización de los cócteles de enzimas para la degradación de la biomasa. Se puede adaptar a las materias primas y varias mezclas de enzimas que contienen múltiples componentes.

Video: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays de polímeros de alto rendimiento para el descubrimiento de Biomateriales

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Investigación

Bioingeniería

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Hidrófobo sal modificada Nafion para inmovilización de enzimas y Estabilización

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Una plataforma automatizada versátil para Micro escala experimentos de estimulación celular

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Desarrollo de una<em&gt; In Vitro</em&gt; Plataforma ocular para probar las lentes de contacto

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Diseño e implementación de un sistema automatizado de Iluminador, cultivo y sistema de muestreo para Aplicaciones microbiana Optogenética

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Una plataforma de pruebas de rendimiento para una conducción Micropump con una placa de electrodo revestido de cobre FR-4

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Protocolo genérico para la optimización de la producción de proteína heteróloga utilizando tecnología Microbioreactor automatizada

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una plataforma de ultra rendimiento microfluidos para la secuenciación del genoma de unicelular

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Una novedosa técnica quirúrgica como base para In Vivo parcial ingeniería hígado de rata

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Reprimiendo la transcripción genética redirigiendo la maquinaria celular con modificadores químicos de epigenéticas

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...