This work was funded in part by the U.S. Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and grant DE-FG02-91ER200021 from the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences. We thank John Scott-Craig and Melissa Borrusch for their material and conceptual contributions.

1. Enzyme Production
<ol>
<li>Produce proteins in Pichia pastoris by cloning of the corresponding genes into vectors pPICZ or pPICZ&#x03B1; and transformation into P. pastoris. Grow cells in 300-ml batches in baffled 500-ml flasks with methanol induction every 24 hr (Banerjee et al., 2010a).</li>
<li>Concentrate and desalt the culture filtrates by tangential flow filtration.</li>
<li>Store the enzymes in small aliquots in 20% glycerol at -80&deg;C. The concentration range is 1-10 mg/ml.</li>
</ol>
2. Design of Experiment
<ol>
<li>Input the number of enzymes to be tested, their upper and lower proportions (e.g. a lower proportion of 5% for core enzymes), and the type of experimental design (e.g., quadratic or cubic) into the Design-Expertsoftware. </li>
<li>Calculate the amount in &mu;g of each enzyme to be added to make a total loading of 15 mg/g glucan, and then calculate the amount of each enzyme in &mu;l to add to each reaction. </li>
<li>Generate Excel worksheets specifying source labware, source wells, destination labware, destination wells, and transfer volumes for dispensing of water and enzymes according to the experimental design and import it into the Biomek FXP software using the transfer-from-file function.</li>
</ol>
3. Enzymatic Hydrolysis
<ol>
<li>Suspend the biomass slurry in 50 mM sodium citrate, pH 4.8, containing 5 &mu;g/ml tetracycline and 5 &mu;g/ml cycloheximide, in the paddle reservoir. </li>
<li>Robotically pipet 200 &mu;l of the slurry into each well of a 96-well deep-well reaction plates using Span-8 1000-&mu;l pipet tips with the last 0.5 cm cut off. Mix the slurry once at 100 &mu;l/sec and then aspirate the same volume from the paddle reservoir and dispense into the plates.</li>
<li>Dispense the enzymes into the same plate using the program entered into the Beckman FX. </li>
<li>Seal the plates with pierceable capmats. </li>
<li>Invert and tap the plates several times to overcome surface tension. Place the plates at 50&deg;C for 48 hr in the hybridization incubator rotating at 10 rpm. </li>
</ol>
4. Determination of Glc and Xyl 
<ol>
<li>Centrifuge the reaction plates for 3 min at 1250 x g in a swinging bucket centrifuge. </li>
<li>Transfer 100 &mu;l of the supernatant into regular 96-well plates using the AP96 pod of the Biomek FX. </li>
<li>Incubate the plates at 90-95&deg;C for 10 min in order to inactivate the enzymes, and then centrifuge at 1250 x g for 30 sec. </li>
<li>For Glc measurements, transfer 12 &mu;l of the supernatants into regular 96-well plates. Add 192 &mu;l of the glucose oxidase/peroxidase (GOPOD) reagent. Incubate the plates at 50&deg;C for 20 min and read the absorbances at 510 nm in a microplate reader. Blank is reaction mixture with no enzyme.</li>
<li>For Xyl measurements, transfer 4 &#x03BC;l into 384-well plates. Add the assay reagents and read the plates at 340 nm. Blank is the reaction mixture with no enzyme.</li>
</ol>
5. Data Analysis
<ol>
<li>Convert the absorbance values to their % Glc and Xyl yields based on the known Glc and Xyl content of the original biomass. Import these percentages as the responses into the Design-Expert software. Check the statistical paramenters to be sure that the criteria for a robust model are fulfilled.</li>
<li>Confirm the best model prediction experimentally.</li>
</ol>
6. Representative Results with GENPLAT: 
(1) Creation of optimized mixtures of individual pure proteins. The results indicate which enzymes are important, and in what proportions, and also which enzymes are not important. Some proteins that are abundant in the T. reesei secretome (Nagendran et al., 2009), such as Cip1 and Cip2, appear to play no role in Glc release from corn stover pretreated by ammonia fiber expansion (AFEX) or alkaline hydrogen peroxide (AHP) (Banerjee et al., 2010b). On the other hand, some proteins are unexpectedly important. For example, endo-xylanases of Glycosyl Hydrolase families 10 and 11 are both important for Glc release; one xylanase cannot substitute for the other (Banerjee et al., 2010b,c). Cel61A, a minor protein in the T. reesei secretome, is very important for Glc but not Xyl release. Some enzymes are important for release of both Glc and Xyl, whereas others are necessary only for one or the other (Banerjee et al., 2010c). 
A synthetic mixture containing 16 components, each at a concentration of 0.94 mg/ml (i.e., a non-optimized mixture), released 38% of the available Glc from AFEX-pretreated DDG. Under identical hydrolysis conditions, an optimized mixture, in which the concentrations of the 16 components ranged from 0% to 32%, released 52% of the Glc (Fig. 2). This experiment illustrates the utility of constructing defined mixtures.
(2) Creation of optimized cocktails for different pretreatment/substrate combinations. Current commercial cellulase preparations are &quot;one-size-fits-all&quot;. In reality, the best cocktail depends on both the substrate and the pretreatment. A single product such as Accellerase 1000 gives different yields from different substrates pretreated in the same way, and from the same substrate exposed to different pretreatments (Fig. 1). We have used GENPLAT to optimize mixtures of pure enzymes for multiple pretreatments and mutiple feedstocks (Banerjee et al., 2010c). Figure 2 shows how the optimal enzyme proportions of 16 pure enzymes differs for AFEX-pretreated corn stover, AHP-pretreated corn stover, and AFEX-pretreated DDG. Only 11 enzymes are shown in Fig. 2 because the optimal proportions of the other five (Cel61B, AbfB, Cip1, Cip2, and Cel12A) were found to be 0% (Banerjee et al., 2010c).
(3) Novel enzyme discovery. The construction of optimized mixtures of pure proteins is an ongoing process. As new proteins become available in pure form (from commercial sources, by heterologous expression, or by purification), they can be systematically tested by adding them to the current optimized set. Novel proteins that contribute to Glc or Xyl release then become part of a new, optimized set. In this way, GENPLAT becomes a platform for the identification of new enzymes for biomass deconstruction. We have made extracts of fungi other than T. reesei and A. niger grown on different substrates. One particular extract boosted Glc yield when added to our core set. The responsible protein was purified and shown to be a novel glycosyl hydrolase not present in any commercial enzyme preparation (Banerjee and Walton, unpublished results).
(4) Discovery of better enzymes. Having shown with GENPLAT which enzymes are most important for degradation of a particular biomass, we have a better understanding of which enzymes should be the focus for improvement. Better examples of the critical enzymes could be found by searching in nature or made by protein engineering. (An enzyme could be &quot;better&quot; than its T. reesei homolog in several ways, depending on the desired property, e.g., higher specific activity, greater synergy, decreased proteolytic sensitivity, or enhanced thermal stability). GENPLAT provides a meaningful platform for assaying potentially better enzymes, because it utilizes a realistically complex cocktail (important because no biomass enzyme works in isolation) and a realistic substrate (important because results with pure cellulose or other artificial substrates may not be relevant to natural lignocellulosic materials).
(5) Commercial enzyme optimization. Sufficiently large quantities of pure enzymes do not yet exist in the real world. Until they do, ethanol producers must rely on commercial products such as Accellerase 1000 and MultifectXylanase. However, mixtures of commercial enzymes generally perform better than any individual one, and GENPLAT can be used to design optimized cocktails of multiple commercial enzymes. Figure 3 shows the use of GENPLAT to optimize a mixture of four commercial enzyme preparations. The optimal proportions for release of Glc from AFEX-pretreated DDG were found to be 47% Accellerase1000, 27% Multifect Pectinase, 22% Novozyme 188, and 4% Multifect Xylanase (Banerjee et al., 2010c). Figure 3B shows the differences in Glc yield from AFEX-DDG with four enzyme preparations; the optimized commercial mixture gives higher Glc yield than Accellerase1000 alone, SpezymeCP alone, or a 16-component synthetic mixture. This experiment also indicates that the 16-component synthetic mixture is missing one or more enzymes needed for optimal Glc release, which are present in one or more of the commercial enzymes. GENPLAT can be used to identify such components.
<img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Figure 1"/> Figure 1. No single commercial enzyme preparation is optimal for all substrates and all pretreatments. Five substrates and three pretreatments were compared under similar conditions of grinding (0.5 mm particle size), enzyme loading (15 mg/g glucan), and hydrolysis conditions (48 hr, 50&deg;C). A. Digestion of different AFEX-pretreated substrates with Accellerase 1000. B. Digestion of corn stover pretreated by three different methods with Accellerase 1000 (see Banerjee et al., 2010c).
<img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Figure 2"/> Figure 2. Optimal proportions of 11 enzymes for release of Glc from AFEX-pretreated corn stover (AFEX-CS), alkaline hydrogen peroxide-pretreated corn stover (AHP-CS), or AFEX-pretreated dried distillers&#x2019; grains (AFEX-DDG). Data are from Banerjee et al. (2010c). Numbers along the top of the bars are the Glc yields as a percent of the total Glc in the biomass sample.
<img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Figure 3"/> Figure 3. A. The use of GENPLAT to produce an optimized mixture of four commercial enzyme preparations for release of Glc from AFEX-pretreated DDG. Multifect Xylanase made the smallest contribution (4%) and was therefore omitted from this ternary diagram. B. Glc yields from AFEX-DDG with different enzyme treatments under identical hydrolysis conditions (15 mg/g glucan enzyme, 48 h, 50&deg;C). &quot;Four-component commercial&quot; has the proportions determined from the experiment shown in 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 conflicts of interest declared.

It is widely recognized that reducing the cost of enzymes is important to the development of an economical lignocellulosic ethanol industry.
Currently available commercial enzyme cocktails are complex and poorly defined mixtures of many proteins (Nagendran et al., 2009), and they are adapted mainly for use on acid-pretreated corn stover. 
In order to accelerate the development of better enzyme cocktails, several laboratories have developed high-throughput platforms for enzyme discovery and characterization. Efforts in this area have incorporated one or more of the following properties also found in GENPLAT: robotic dispensing of enzymes and biomass slurries, statistical design of experiment, and/or automated determination of Glc and Xyl (Berlin et al., 2007; Decker et al., 2009; Kim et al., 1998; King et al., 2009). GENPLAT extends these earlier efforts, most significantly in the complexity of the enzyme mixtures that can be analyzed from at most 6 components in the earlier studies to more than 16 in our latest work (Banerjee et al., 2010c). Additional key features of GENPLAT are the use of a bead mixing chamber (paddle reservoir) that can keep stover slurries suspended during dispensing; gentle mixing during digestion by end-over-end rotation; and automated colorimetric determination of Glu and Xyl.

Equipments and software used:
<ol>
<li>Design&#x2013;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 laboratory automation workstation (Beckman-Coulter, Fullerton, CA)</li>
<li>Biomek FXP software, Version 3.3 (Beckman-Coulter, Fullerton, CA)</li>
<li>Paddle reservoir (Model VP 756C-1P100, V &amp; P Scientific Inc., San Diego)</li>
<li>Plates, 96-deep (1.5-ml) well (Abgene, Fisher Scientific, Epson, UK)</li>
<li>Pierceable capmats (USA Scientific, Ocala, FL)</li>
<li>Rotating hybridization incubator (Model 5420,VWR)</li>
<li>Swinging-bucket centrifuge (Model 5810R, Eppendorf, Hamburg, Germany)</li>
<li>Plates, Costar 96-well (Abgene, Fisher Scientific, Epson, UK)</li>
<li>Plates, 384-well (BD Falcon, BD Biosciences, San Jose, CA)</li>
</ol>
Reagents used:
<ol>
<li>Pichia pastoris and vectors: strain X33, and pPICZ and pPICZ&alpha; (Invitrogen, Carlsbad, CA)</li>
<li>T. reesei and vectors: strain QM9414 (US Department of Agriculture National Center for Agricultural Utilization Research, Peoria, IL), and pCB1004-EV (Banerjee et al., 2010b)</li>
<li>Accellerase 1000 (lot number 1600844643) (Genencor, Inc., Rochester, NY)</li>
<li>50 mM sodium citrate buffer, pH 4.8</li>
<li>Tetracycline stock: 10 mg/ml </li>
<li>Cycloheximide stock: 10 mg/ml </li>
<li>GOPOD assay kit , catalog K-GLUC (Megazyme, Bray, Ireland)</li>
<li>Xylose assay kit, catalog K-XYLOSE (Megazyme, Bray, Ireland)</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).
<div id="myEventWatcherDiv">
	 </div>

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 (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

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

15.4K Aufrufe.  Michigan State University. GENPLAT (GLBRC Enzyme Platform) ist eine automatisierte Plattform für die Entdeckung und Optimierung von Enzym-Cocktails für Biomasse Abbau. Es kann zu mehreren Rohstoffen und Mischungen von Enzymen, die mehrere Komponenten angepasst werden.

Serie: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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