Questo lavoro è stato finanziato in parte dal Dipartimento dell&#39;Energia degli Stati Uniti dei Grandi Laghi Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) e concedere DE-FG02-91ER200021 da parte del Dipartimento dell&#39;Energia degli Stati Uniti, Ufficio di Basic Sciences Energia, Divisione di Scienze Chimiche, Scienze geologiche e Bioscienze. Ringraziamo John Scott-Craig e Melissa Borrusch per il loro materiale e contributi concettuali.

1. Enzima di produzione <ol><li> Producono proteine ​​in Pichia pastoris per la clonazione dei geni corrispondenti in vettori pPICZ o pPICZα e la trasformazione in P. pastoris. Crescere le cellule in lotti da 300 ml in sconcertato da 500 ml palloni con induzione metanolo ogni 24 ore (Banerjee et al. 2010a). </li><li> Concentrato e desalt i filtrati dalla cultura filtrazione a flusso tangenziale. </li><li> Conservare gli enzimi in piccole aliquote nel 20% glicerolo a -80 ° C. L&#39;intervallo di concentrazione è 1-10 mg / ml. </li></ol> 2. Progettazione di Experiment <ol><li> Inserire il numero di enzimi da testare, i loro superiori e inferiori proporzioni (ad esempio una percentuale inferiore al 5% per gli enzimi core), e il tipo di disegno sperimentale (ad esempio, quadratica o cubica) nella progettazione Expertsoftware. </li><li> Calcolare la quantità in mg di ciascun enzima da aggiungere per fare un carico totale di 15 mg / g di glucano, e quindi calcolare la quantità di ciascun enzima in microlitri da aggiungere allaogni reazione. </li><li> Generare fogli di lavoro Excel specificando da laboratorio sorgente, pozzi fonte, da laboratorio di destinazione, pozzi di destinazione e dei volumi di trasferimento per l&#39;erogazione di acqua ed enzimi secondo il disegno sperimentale e importarlo nel software Biomek FXP utilizzando il trasferimento di file funzione. </li></ol> 3. Idrolisi enzimatica <ol><li> Sospendere il liquame biomassa in 50 mM di sodio citrato, pH 4.8, contenente 5 mg / ml di tetraciclina e 5 mg / ml cicloeximide, nel serbatoio pagaia. </li><li> Roboticamente pipetta 200 ul del liquame in ciascun pozzetto di un pozzo profondo 96-pozzetti di reazione mediante Span-8 1000-microlitri puntali con gli ultimi 0,5 centimetri tagliato. Mescolare l&#39;impasto una volta a 100 l / sec e quindi aspirare lo stesso volume dal serbatoio pagaia e distribuire nei piatti. </li><li> Erogare gli enzimi nella piastra stessa utilizzando il programma inserito nel FX Beckman. </li><li> Sigillare le piastre con capmats perforabile. </li><li> Inverti e toccarele piastre più volte di superare la tensione superficiale. Posizionare le piastre a 50 ° C per 48 ore nella ibridazione rotazione incubatore a 10 giri. </li></ol> 4. Determinazione del Glc e Xyl <ol><li> Centrifugare le piastre di reazione per 3 min a 1250 xg in una centrifuga secchio oscillante. </li><li> Trasferire 100 ml di surnatante in regolare piastre con 96 pozzetti utilizzando l&#39;AP96 pod della Biomek FX. </li><li> Incubare le piastre a 90-95 ° C per 10 minuti per inattivare gli enzimi, quindi si centrifuga a 1250 xg per 30 sec. </li><li> Per le misure Glc, trasferire 12 microlitri della surnatanti in regolare piastre a 96 pozzetti. Aggiungere 192 microlitri della ossidasi / perossidasi del glucosio (goPod) reagente. Incubare le piastre a 50 ° C per 20 minuti e leggere le assorbanze a 510 nm in un lettore di micropiastre. Blank è miscela di reazione senza enzima. </li><li> Per le misure Xyl, trasferimento 4 microlitri in piastre da 384 pozzetti. Aggiungere i reagenti e leggere le piastre a 340 nm. Vuotoè la miscela di reazione senza enzima. </li></ol> 5. Analisi dei dati <ol><li> Convertire i valori di assorbanza% Glc loro e rese Xyl basato sul noto Glc e contenuti Xyl della biomassa originale. Importare queste percentuali delle risposte in Design-Expert. Controllare la paramenters statistiche per essere sicuri che i criteri per un robusto modello sono soddisfatte. </li><li> Confermare la previsione miglior modello sperimentale. </li></ol> 6. Risultati rappresentante con GENPLAT: (1) La creazione di miscele ottimizzata di singole proteine ​​pure. I risultati indicano che gli enzimi sono importanti, e in quali proporzioni, e anche che gli enzimi non sono importanti. Alcune proteine ​​che sono abbondanti nel T. reesei secretome (Nagendran et al., 2009), come Cip1 e Cip2, sembrano svolgere alcun ruolo nel Glc comunicato dal mais pretrattati con espansione in fibra di ammoniaca (AFEX) o alcaline hyd Stover<html> Rogen perossido (AHP) (Banerjee et al. 2010b). D&#39;altra parte, alcune proteine ​​sono inaspettatamente importanti. Per esempio, endo-xilanasi delle famiglie glicosil idrolasi 10 e 11 sono entrambi importanti per Glc rilascio, uno xilanasi non può sostituire l&#39;altro (Banerjee et al, 2010b, c).. Cel61A, una proteina minore del T. reesei secretome, è molto importante per Glc ma non Xyl rilascio. Alcuni enzimi sono importanti per il rilascio di entrambi i Glc e Xyl, mentre altri sono necessarie solo per una o l&#39;altra (Banerjee et al. 2010C). Una miscela di sintesi contenente 16 componenti, ciascuna ad una concentrazione di 0,94 mg / ml (cioè un non ottimizzato miscela), rilasciata il 38% dei disponibili Glc da AFEX pre-trattati con DDG. In condizioni di idrolisi identiche, una miscela ottimizzata, in cui le concentrazioni dei 16 componenti varia da 0% al 32%, rilasciata il 52% del Glc (Fig. 2). Questo esperimento illustra l&#39;utilità della costruzione di miscele definite. _content &quot;&gt; (2) La creazione di cocktail ottimizzata per il pretrattamento diversi / combinazioni substrato. attuali preparazioni cellulasi commerciali sono&quot; one-size-fits-all &quot;. In realtà, i migliori cocktail dipende sia dal substrato e il pretrattamento. Un unico prodotto come Accellerase 1000 dà rese diverse da diversi substrati pretrattati allo stesso modo, e dal substrato stesso esposto a pretrattamenti (Fig. 1). Abbiamo usato GENPLAT per ottimizzare le miscele di enzimi per puro pre-trattamenti multipli e materie prime mutiple (Banerjee et al. 2010C). Figura 2 mostra come le proporzioni degli enzimi ottimale di 16 enzimi puro si distingue per paglia del mais AFEX-pretrattati, AHP-pretrattati paglia del mais, e AFEX pre-trattati con DDG. Solo 11 gli enzimi sono mostrati in fig. 2 perché l&#39;ottimale proporzioni degli altri cinque (Cel61B, AbfB, Cip1, Cip2 e Cel12A) sono risultate pari a 0% (Banerjee et al. 2010C). (3) Novel scoperta degli enzimi </strong&gt;. La costruzione di miscele ottimizzata di proteine ​​pure è un processo continuo. Come si rendono disponibili nuove proteine ​​in forma pura (di origine commerciale, di espressione eterologa, o purificazione), possono essere sistematicamente testati aggiungendoli al set corrente ottimizzato. Nuove proteine ​​che contribuiscono a Glc o Xyl rilasciare poi diventare parte di una nuova serie ottimizzata. In questo modo, GENPLAT diventa una piattaforma per l&#39;identificazione di nuovi enzimi per la decostruzione della biomassa. Abbiamo fatto di estratti di funghi diverso da T. reesei e A. niger cresciuti su diversi substrati. Un estratto di particolare potenziato resa Glc quando aggiunto al nostro set base. La proteina responsabile era purificata e dimostrato di essere un romanzo glicosil idrolasi non presente in alcun preparato enzimatico commerciale (Banerjee e Walton, risultati non pubblicati). (4) La scoperta di una migliore enzimi. Dopo aver dimostrato con GENPLAT che gli enzimi sono più importanti per la degradazione di unparticolare la biomassa, abbiamo una migliore comprensione di enzimi che dovrebbe essere l&#39;obiettivo di miglioramento. Esempi migliori degli enzimi critica potrebbe essere quella di cercare in natura o resi mediante ingegneria proteica. (Un enzima potrebbe essere &quot;migliore&quot; rispetto al suo omologo T. reesei in diversi modi, a seconda della proprietà desiderata, ad esempio, l&#39;attività più specifiche, una maggiore sinergia, diminuita sensibilità proteolitici, o maggiore stabilità termica). GENPLAT fornisce una piattaforma significativa per saggiare gli enzimi potenzialmente migliore, perché utilizza un cocktail realisticamente complessi (importante perché nessuna enzima biomassa lavora in isolamento) e un substrato realistico (importante perché i risultati con pura cellulosa o altri substrati artificiali non possono essere rilevanti per lignocellulosici naturali materiali). (5) ottimizzazione enzima commerciale. Quantità sufficientemente grande di enzimi puri non esistono ancora nel mondo reale. Finché non lo farà, l&#39;etanolo proproduttori devono fare affidamento su prodotti commerciali come Accellerase 1000 e MultifectXylanase. Tuttavia, miscele di enzimi commerciali in genere un rendimento migliore rispetto a qualsiasi individuo, e GENPLAT può essere usato per progettare cocktail ottimizzato di più enzimi commerciale. La figura 3 mostra l&#39;uso di GENPLAT per ottimizzare una miscela di quattro preparati enzimatici commerciali. Le proporzioni ottimali per il rilascio di Glc da AFEX pre-trattati con DDG sono risultate essere il 47% Accellerase1000, 27% Multifect Pectinasi, 22% Novozyme 188, e il 4% Multifect Xylanase (Banerjee et al. 2010C). Figura 3B mostra le differenze di rendimento Glc da AFEX-DDG con quattro preparati enzimatici, la miscela ottimizzata commerciale dà maggiore rendimento rispetto Glc Accellerase1000 solo, SpezymeCP da solo, o un 16-componente miscela sintetica. Questo esperimento indica anche che l&#39;16-componente miscela sintetica non è presente uno o più enzimi necessari per il rilascio ottimale Glc, che sono presenti in uno o più degli enzimi commerciale. GENPLAT ci può essereed per identificare i componenti del genere. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Figura 1"/> Figura 1. Nessuna preparazione singolo enzima commerciale è ottimale per tutti i sottofondi e tutti i pre-trattamenti. Cinque substrati e tre pre-trattamenti sono stati confrontati in condizioni simili di macinazione (0,5 granulometria mm), enzima di carico (15 mg / g glucani), e le condizioni di idrolisi (48 ore, 50 ° C). A. digestione di diverse AFEX-substrati pretrattati con Accellerase 1000. Digestione B. di paglia del mais pretrattati con tre metodi diversi con Accellerase 1000 (vedi Banerjee et al. 2010C). <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Figura 2"/> Figura 2. Proporzione ottimale di 11 enzimi per il rilascio del Glc da AFEX-pretrattati paglia del mais (AFEX-CS), perossido di idrogeno alcalino-pretrattati paglia del mais (AHP-CS), o AFEX-pretrattati secca distillaregrani ERS &#39;(AFEX-DDG). I dati sono da Banerjee et al. (2010C). Numeri lungo la parte superiore delle barre sono le rese Glc come percentuale del totale Glc nel campione biomassa. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Figura 3"/> Figura 3. A. L&#39;uso di GENPLAT per produrre una miscela ottimizzata di quattro preparati enzimatici commerciali per il rilascio di Glc da AFEX pre-trattati con DDG. Multifect Xylanase fatto il più piccolo contributo (4%) ed è stato quindi omesso da questo diagramma ternario. B. rendimenti Glc da AFEX-DDG con trattamenti enzimatici diversi idrolisi in condizioni identiche (15 mg / g enzima glucano, 48 h, 50 ° C) . &quot;Quattro-componente commerciale&quot; ha le proporzioni stabilite dal l&#39;esperimento mostrato in fig. 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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Nessun conflitto di interessi dichiarati.

E &#39;ampiamente riconosciuto che la riduzione del costo di enzimi è importante per lo sviluppo di un settore economico di etanolo lignocellulosiche. Attualmente sono disponibili cocktail di enzimi commerciali sono miscele complesse e scarsamente definita di molte proteine ​​(Nagendran et al., 2009), e si adattano soprattutto per l&#39;uso su pre-trattati con acido paglia del mais. Al fine di accelerare lo sviluppo di cocktail di enzimi meglio, diversi laboratori hanno sviluppato piattaforme high-throughput per la scoperta e la caratterizzazione degli enzimi. Sforzi in questo campo hanno incorporato una o più delle seguenti proprietà anche in GENPLAT: erogazione robotica di enzimi e fanghi di biomassa, il modello statistico di sperimentazione, e / o determinazione automatica di Glc e Xyl (Berlin et al, 2007; Decker et. al, 2009;. Kim et al, 1998;. King et al, 2009).. GENPLAT estende questi sforzi in precedenza, la maggior parte in modo significativo nella complessità delle miscele enzima che può essere analizzata da un massimo di 6 componenti ini primi studi a oltre 16 nel nostro ultimo lavoro (Banerjee et al. 2010C). Altre caratteristiche chiave del GENPLAT sono l&#39;uso di una camera di miscelazione tallone (serbatoio paddle), che può mantenere fanghi paglia sospesi durante l&#39;erogazione, la miscela dolce durante la digestione entro la fine del-over-end di rotazione; e automatizzato determinazione colorimetrica di Glu e Xyl.

Apparecchiature e software utilizzati: <ol><li> Design-Expert, versione 8.0 (Stat-Ease, Inc., Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP laboratorio di automazione workstation (Beckman-Coulter, Fullerton, CA) </li><li> Biomek FXP software, Versione 3.3 (Beckman-Coulter, Fullerton, CA) </li><li> Pale serbatoio (modello VP-756C 1P100, V &amp; P Scientific Inc., San Diego) </li><li> Piatti, 96-profondo (1,5 ml) e (Abgene, Fisher Scientific, Epson, UK) </li><li> Capmats perforabile (USA scientifico, Ocala, FL) </li><li> Rotazione ibridazione incubatore (modello 5420, VWR) </li><li> Swinging-bucket centrifuga (Model 5810R, Eppendorf, Amburgo, Germania) </li><li> Piatti, Costar a 96 pozzetti (Abgene, Fisher Scientific, Epson, UK) </li><li> Piatti, da 384 pozzetti (BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Reagenti utilizzati: <ol><li> Pichia pastoris e vettori: tensione X33, e pPICZ e pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei e vettori: ceppo QM9414 (US Department of Agriculture Centro Nazionale per la Ricerca Agricola utilizzo, Peoria, IL), e pCB1004-EV (Banerjee et al, 2010b). </li><li> Accellerase 1000 (numero di lotto 1600844643) (Genencor, Inc., Rochester, NY) </li><li> 50 mM tampone citrato di sodio, pH 4,8 </li><li> Tetraciclina magazzino: 10 mg / ml </li><li> Cicloeximide magazzino: 10 mg / ml </li><li> GoPod test kit, catalogo K-Gluc (Megazyme, Bray, Irlanda) </li><li> Xilosio test kit, catalogo K-XYLOSE (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) è una piattaforma automatizzata per la scoperta e l&#39;ottimizzazione di cocktail di enzimi per la degradazione della biomassa. Può essere adattato alle materie prime più e miscele di enzimi contenenti più componenti.

GENPLAT: una piattaforma automatizzata per la scoperta degli enzimi biomassa e ottimizzazione 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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Bioengineering

15.3K Views.  Michigan State University. GENPLAT (Enzyme GLBRC Platform) è una piattaforma automatizzata per la scoperta e l'ottimizzazione di cocktail di enzimi per la degradazione della biomassa. Può essere adattato alle materie prime più e miscele di enzimi contenenti più componenti.

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 polimerici per Discovery High Throughput di Biomateriali

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

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Bioingegneria

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.

Idrofobo Salt-modificato Nafion per l&#39;immobilizzazione degli enzimi e la stabilizzazione

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 piattaforma automatizzata versatile per micro-scala Cellulari esperimenti di stimolazione

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.

Sviluppo di un<em&gt; In Vitro</em&gt; Oculare piattaforma per testare Lenti a contatto

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.

Progettazione e Realizzazione di un Illuminating automatizzato, Coltura, e di campionamento per microbica optogenetic Applicazioni

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 piattaforma di test delle prestazioni per una conduzione Micropump con una piastra elettrodo rivestito di rame 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.

Protocollo generico per l'ottimizzazione della produzione di proteine eterologhe utilizzando tecnologia automatizzata di 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.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

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 nuova tecnica chirurgica come una Fondazione per In Vivo parziale ingegneria del fegato nel ratto

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.

Reprimere la trascrizione genica reindirizzando macchinario cellulare con modificatori epigenetici chimici

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