Este trabalho foi financiado em parte pelo Departamento de Energia dos EUA Grande Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) e conceder-DE-FG02 91ER200021 do Departamento de Energia dos EUA, Escritório de ciências básicas da energia, Divisão de Ciências Químicas, Geociências e Biociências. Agradecemos a John Scott-Craig e Melissa Borrusch para seus materiais e contribuições conceituais.

1. Produção da enzima <ol><li> Produzir proteínas em Pichia pastoris por clonagem dos genes correspondentes em vetores pPICZ ou pPICZα e transformação em P. pastoris. Cultivar células em 300 ml em lotes perplexo de 500 ml frascos com a indução de metanol cada hr 24 (Banerjee et al., 2010a). </li><li> Concentrar e dessalinizar os filtrados de cultura por filtração de fluxo tangencial. </li><li> Armazenar as enzimas em pequenas alíquotas em glicerol 20% a -80 ° C. A faixa de concentração é 1-10 mg / ml. </li></ol> 2. Design da Experiência <ol><li> Introduza o número de enzimas a serem testados, o seu superior e proporções mais baixas (por exemplo, uma menor proporção de 5% para enzimas core), e do tipo de projeto experimental (por exemplo, quadrática ou cúbica) para o Design-Expertsoftware. </li><li> Calcular a quantidade em mg de cada enzima para ser adicionado ao fazer uma carga total de 15 glucan mg / g, e depois calcular a quantidade de cada enzima em mL para adicionarcada reação. </li><li> Gerar planilhas do Excel especificando labware fonte, poços fonte, material de laboratório de destino, poços de destino, e os volumes de transferência para distribuição de água e enzimas de acordo com o delineamento experimental e importá-lo para o software FXP Biomek usando a função de transferência-do-arquivo. </li></ol> 3. A hidrólise enzimática <ol><li> Suspender a suspensão de biomassa em 50 mM de citrato de sódio, pH 4,8, contendo 5 mg / ml de tetraciclina e 5 cicloheximida mcg / ml, no reservatório de remo. </li><li> Roboticamente pipeta 200 mL da suspensão em cada poço de uma placas de 96 poços profundos bem-Span reação utilizando-8 dicas 1000-Pipete com os últimos 0,5 centímetros cortada. Misture a pasta uma vez em 100 l / seg e depois aspirar o mesmo volume do reservatório paddle e dispensar para as placas. </li><li> Dispense as enzimas para a mesma placa usando o programa entrou no FX Beckman. </li><li> Selar as placas com capmats perfurável. </li><li> Inverter e toqueas placas diversas vezes para vencer a tensão superficial. Coloque as placas a 50 ° C por 48 h em incubadora a hibridização girando a 10 rpm. </li></ol> 4. Determinação de Glc e XYL <ol><li> Centrifugar as placas de reação para 3 min a 1250 xg em uma centrífuga de caçamba móvel. </li><li> Transferência de 100 ml do sobrenadante para regulares placas de 96 poços usando o pod AP96 do FX Biomek. </li><li> Incubar as placas a 90-95 ° C por 10 min, a fim de inativar as enzimas, e, em seguida, centrifugar a 1250 xg por 30 seg. </li><li> Para medições Glc, transferir 12 mL do sobrenadante em regulares placas de 96 poços. Adicionar 192 mL do reagente glicose oxidase / peroxidase (GOPOD). Incubar as placas a 50 ° C por 20 min e ler as absorvâncias a 510 nm em um leitor de microplacas. Em branco é mistura de reação sem enzima. </li><li> Para medições XYL, transferência de 4 mL em 384 poços pratos. Adicionar os reagentes de ensaio e ler as placas a 340 nm. Em brancoé a mistura de reação sem enzima. </li></ol> 5. Análise de Dados <ol><li> Converter os valores de absorbância ao seu Glc% e os rendimentos XYL baseado no Glc conhecido e conteúdo XYL da biomassa original. Importar esses percentuais como as respostas para o software de Design-Expert. Verifique o paramenters estatísticos a certeza de que os critérios para um modelo robusto são cumpridas. </li><li> Confirmar a previsão de melhor modelo experimental. </li></ol> 6. Resultados representante com GENPLAT: (1) Criação de misturas otimizadas de proteínas individuais puro. Os resultados indicam que as enzimas são importantes, e em que proporções, e também quais as enzimas não são importantes. Algumas proteínas que são abundantes na T. reesei secretome (Nagendran et al., 2009), como CIP1 e CIP2, parecem desempenhar nenhum papel na libertação de Glc palha de milho pré-tratados pela expansão de amônia fibra (AFEX) ou alcalina hyd<html> rogen peróxido (AHP) (Banerjee et al., 2010b). Por outro lado, algumas proteínas são inesperadamente importante. Por exemplo, endo-xilanases de famílias hydrolase glicosil 10 e 11 são ambos importantes para Glc libertação; uma xilanase não pode substituir o outro (Banerjee et al, 2010b, c).. Cel61A, uma proteína menor no T. reesei secretome, é muito importante para Glc mas não XYL lançamento. Algumas enzimas são importantes para a liberação de ambos os Glc e XYL, enquanto outras são necessárias apenas para um ou outro (Banerjee et al., 2010C). A mistura sintética contendo 16 componentes, cada um com uma concentração de 0,94 mg / ml (ou seja, uma mistura não-otimizado), lançado 38% do Glc disponível a partir de pré-tratamento AFEX DDG. Em condições idênticas de hidrólise, uma mistura optimizada, em que as concentrações dos 16 componentes variou de 0% a 32%, liberado 52% do Glc (Fig. 2). Este experimento ilustra a utilidade da construção de misturas definidas. _content &quot;&gt; Criação (2) de cocktails otimizado para pré-tratamento diferente / combinações de substrato. atual celulase preparações comerciais são&quot; one-size-fits-all &quot;. Na realidade, o melhor cocktail depende tanto do substrato e pré-tratamento. Um produto único como Accellerase 1000 dá rendimentos diferentes dos diferentes substratos pré-tratados da mesma forma, e do mesmo substrato exposto a diferentes pré-tratamentos (Fig. 1). Usámos GENPLAT para otimizar misturas de enzimas pré-tratamentos puro para múltiplas e matérias-primas mutiple (Banerjee et al., 2010C). Figura 2 mostra como as proporções de enzima ideal de 16 enzimas pura difere para AFEX pré-tratamento palha de milho, AHP pré-tratamento palha de milho, e AFEX pré-tratamento DDG. Apenas 11 enzimas são mostrados na Figura 2. porque o ideal proporções dos outros cinco (Cel61B, AbfB, CIP1, CIP2 e Cel12A) foram encontrados para ser 0% (Banerjee et al., 2010C). (3) a descoberta de enzimas Novel </strong&gt;. A construção de misturas otimizadas de proteínas pura é um processo contínuo. Como novas proteínas tornam-se disponíveis na forma pura (a partir de fontes comerciais, por expressão heteróloga, ou purificação), eles podem ser sistematicamente testados, adicionando-os ao conjunto atual otimizada. Novas proteínas que contribuem para Glc ou XYL release então tornar-se parte de um conjunto novo e otimizado. Desta forma, torna-se GENPLAT uma plataforma para a identificação de novas enzimas para a desconstrução da biomassa. Fizemos extratos de outros fungos que T. reesei e A. niger cultivado em diferentes substratos. Um extrato particular impulsionou o rendimento Glc quando adicionado ao nosso conjunto central. A proteína responsável foi purificada e demonstrou ser um romance glicosil hidrolase não está presente em qualquer preparação enzimática comercial (Banerjee e Walton, resultados não publicados). (4) A descoberta de enzimas melhor. Tendo mostrado com GENPLAT enzimas que são mais importantes para a degradação de umabiomassa particular, temos uma melhor compreensão de quais enzimas deve ser o foco para a melhoria. Melhores exemplos das enzimas crítica pode ser encontrada através de pesquisa na natureza ou obtidos por engenharia de proteínas. (Uma enzima poderia ser &quot;melhor&quot; do que o seu homólogo T. reesei de várias maneiras, dependendo da propriedade desejada, atividade, por exemplo, mais específicos, maior sinergia, diminuição da sensibilidade proteolítica, ou estabilidade térmica melhorada). GENPLAT fornece uma plataforma significativa para a dosagem de enzimas potencialmente melhor, porque ele utiliza um cocktail realisticamente complexas (importante porque nenhuma enzima biomassa funciona isoladamente) e um substrato realista (importante porque os resultados com celulose pura ou outros substratos artificiais não podem ser relevantes para lignocelulósicos naturais materiais). (5) otimização da enzima comercial. Quantidades suficientemente grande de enzimas puras ainda não existem no mundo real. Até que o façam, o etanol protores deve confiar em produtos comerciais, como Accellerase 1000 e MultifectXylanase. No entanto, as misturas de enzimas comerciais geralmente têm melhor desempenho do que qualquer um indivíduo, e GENPLAT pode ser usada para projetar cocktails otimizado de várias enzimas comerciais. A Figura 3 mostra o uso de GENPLAT para otimizar uma mistura de quatro preparações de enzimas comerciais. As proporções ideais para a liberação de Glc AFEX de pré-tratamento DDG foram encontrados para ser 47% Accellerase1000, 27% Multifect Pectinase, 22% Novozyme 188, e 4% Multifect xilanase (Banerjee et al., 2010C). Figura 3B mostra as diferenças no rendimento de AFEX Glc-DDG com quatro preparações enzimáticas; a mistura otimizada comerciais dá maior rendimento do que Glc Accellerase1000 sozinho, SpezymeCP sozinho, ou uma mistura de 16 componentes sintéticos. Esta experiência também indica que a mistura de 16 componentes sintéticos está faltando uma ou mais enzimas necessárias para uma ótima Glc lançamento, que estão presentes em uma ou mais das enzimas comerciais. GENPLAT pode ser-nosed para identificar tais componentes. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Figura 1"/> Figura 1. Nenhuma preparação única enzima comercial é ideal para todos os substratos e todos os pré-tratamentos. Cinco substratos e três pré-tratamentos foram comparados em condições similares de moagem (0,5 milímetros de tamanho de partículas), enzima de carga (15 mg glucana / g), e as condições de hidrólise (48 hr, 50 ° C) Digestão. A. de diferentes substratos AFEX pré-tratamento com Accellerase 1000. B. A digestão de palha de milho pré-tratados por três métodos diferentes, com Accellerase 1000 (ver Banerjee et al., 2010C). <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Figura 2"/> Figura 2. Proporções Optimal de 11 enzimas para liberação de Glc AFEX de pré-tratamento palha de milho (AFEX-CS), peróxido de hidrogênio alcalino pré-tratamento palha de milho (AHP-CS), ou pré-tratamento AFEX secas destilargrãos ers &quot;(AFEX-DDG). Os dados são do Banerjee et al. (2010C). Números ao longo do topo das barras são os rendimentos Glc como uma porcentagem do Glc total na amostra de biomassa. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Figura 3"/> Figura 3. A. O uso de GENPLAT para produzir uma mistura optimizada de quatro preparações de enzimas comerciais para liberação de Glc AFEX de pré-tratamento DDG. Multifect xilanase fez a menor contribuição (4%) e, portanto, omitidos neste diagrama ternário. Rendimentos B. Glc de AFEX-DDG com tratamentos diferentes enzimas em condições idênticas de hidrólise (15 enzima glucano mg / g, 48 h, 50 ° C) . &quot;Four-componente comercial&quot; tem as proporções determinadas a partir do experimento mostrado na figura. 3A.

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	<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. 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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. 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Prog. 23, 1270-1276 (2007).</li></ol>

Não há conflitos de interesse declarados.

É amplamente reconhecido que a redução do custo das enzimas é importante para o desenvolvimento de uma indústria de etanol lignocelulósico econômica. Atualmente disponíveis coquetéis enzimáticos comerciais são misturas complexas e mal definidas de muitas proteínas (Nagendran et al., 2009), e eles são adaptados principalmente para uso em pré-tratamento ácido palha de milho. A fim de acelerar o desenvolvimento de cocktails melhor enzima, vários laboratórios têm desenvolvido high-throughput plataformas para a descoberta e caracterização de enzimas. Esforços nesta área têm incorporado um ou mais das seguintes propriedades também encontrada em GENPLAT: dispensa robótico de enzimas e suspensões de biomassa, a concepção estatística de experimento, e / ou determinação automatizada de Glc e XYL (Berlin et al, 2007; Decker et. al, 2009;. Kim et al, 1998;. King et al, 2009).. GENPLAT estende esses esforços anteriores, mais significativamente na complexidade das misturas de enzimas que podem ser analisados ​​a partir de no máximo seis componentes emos estudos mais cedo para mais de 16 no nosso mais recente trabalho (Banerjee et al., 2010C). Adicionais das principais características do GENPLAT são o uso de uma câmara de talão de mistura (reservatório de paddle) que pode manter pastas suspensas durante stover dispensação; suave mistura durante a digestão até o final de over-final da rotação, e determinação colorimétrica automatizada de Glu e XYL.

Equipamentos e software utilizados: <ol><li> Design-Expert software, Version 8.0 (Stat-Ease, Inc., Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP laboratório de automação de estações de trabalho (Beckman-Coulter, Fullerton, CA) </li><li> Biomek FXP software, Version 3.3 (Beckman-Coulter, Fullerton, CA) </li><li> Reservatório de Paddle (Modelo VP-756C 1P100, V &amp; P Scientific Inc., San Diego) </li><li> Placas, 96 de profundidade (1,5 ml) bem (Abgene, Fisher Scientific, Epson, UK) </li><li> Capmats perfurável (EUA Científico, Ocala, FL) </li><li> Hibridização rotativa incubadora (Modelo 5420, VWR) </li><li> Balançando-balde de centrifugação (Modelo 5810R, Eppendorf, Hamburgo, Alemanha) </li><li> Placas, Costar de 96 poços (Abgene, Fisher Scientific, Epson, UK) </li><li> Placas, de 384 poços (BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Reagentes utilizados: <ol><li> Pichia pastoris e vetores: cepa X33, e pPICZ e pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei e vetores: tensão QM9414 (Departamento de Agricultura dos EUA Centro Nacional de Pesquisa de Utilização Agrícola, Peoria, IL), e pCB1004-EV (Banerjee et al, 2010b). </li><li> Accellerase 1000 (número do lote 1600844643) (Genencor, Inc., Rochester, NY) </li><li> 50 mM tampão citrato de sódio, pH 4,8 </li><li> Tetraciclina estoque: 10 mg / ml </li><li> Cicloheximida estoque: 10 mg / ml </li><li> GOPOD ensaio kit, catálogo K-glic (Megazyme, Bray, Irlanda) </li><li> Xilose ensaio kit, catálogo K-xilose (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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GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT (Enzyme GLBRC Platform) é uma plataforma automatizada para a descoberta e otimização de coquetéis enzimáticos para a degradação da biomassa. Ele pode ser adaptado para matérias-primas múltiplas e misturas de enzimas contendo vários componentes.

GENPLAT: uma plataforma automatizada para Discovery Biomassa Enzima e Otimização Cocktail

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

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15.3K visualizações.  Michigan State University. GENPLAT (Enzyme GLBRC Platform) é uma plataforma automatizada para a descoberta e otimização de coquetéis enzimáticos para a degradação da biomassa. Ele pode ser adaptado para matérias-primas múltiplas e misturas de enzimas contendo vários componentes.

Vídeo: 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 para Discovery High Throughput de Biomateriais

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

Bioengenharia

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.

Nafion-Sal modificado hidrofóbico para imobilização de enzimas e Estabilização

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.

A plataforma automatizada versátil para experiências de estimulação Micro escala celulares

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.

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ocular Platform para testar as lentes de contato

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.

Design e Implementação de um Automated Illuminating, A cultura e sistema de amostragem para Aplicações Microbial 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.

Uma plataforma de testes de desempenho para uma microbomba de condução com uma placa de eletrodos de cobre-folheada 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 otimização da produção de proteína heteróloga usando tecnologia automatizada de Microbioreactor

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.

Uma plataforma Microfluidic de ultra alta taxa de transferência para o sequenciamento do genoma de célula única

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.

Uma nova técnica cirúrgica como fundamento na Vivo parcial engenharia fígado de rato

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.

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

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