This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.

1. Preparing the Metagenomic Library with pNP-GESS
<ol>
	<li>Construct a metagenomic library in E. coli with a fosmid vector using a fosmid library production kit according to the manufacturer&#39;s protocol 5.</li>
	<li>Aliquot 100 &#181;l of the library for storage at &#8722;70 &#176;C, which is a source of metagenomic library cells. 
	Note: The optical density of a sample measured at a wavelength of 600 nm (OD600) of this library stock is approximately 100.</li>
	<li>Thaw 100 &#181;l of the stock metagenomic library on ice and inoculate in a 500 ml flask containing 50 ml Luria-Bertani (LB) and 12.5 &#181;g/ml chloramphenicol followed by 37 &#176;C incubation for 2 hr.</li>
	<li>Harvest the cells in a 50 ml conical tube by centrifugation at 1,000 x g for 20 min at 4 &#176;C.</li>
	<li>Resuspend the pellet quickly in 50 ml ice-cold distilled water (DW) and centrifuge at 1,000 x g for 20 min at 4 &#176;C again.</li>
	<li>Resuspend the pellet in 50 &#181;l ice-cold DW with 10% (v/v) glycerol. Use 50 &#181;l of this cell aliquot for electroporation.</li>
	<li>Place the mixture of electrocompetent cells 50 &#181;l and pGESS(E135K)5 DNA (100ng) in an ice-cold electroporation cuvette and electroporate (1.8 kV/cm, 25 &#181;F) the mixture.</li>
	<li>Quickly add 1 ml super optimal broth with catabolite repression (SOC) medium and resuspend the cells gently.</li>
	<li>Transfer the cells into 14 ml round-bottom tube using a pipette and incubate at 37 &#176;C for 1 hr.</li>
	<li>Spread 500 &#181;l of the recovered cells on a LB 20 x 20 cm square plate containing 12.5 &#181;g/ml chloramphenicol and 50 &#181;g/ml ampicillin. Incubate plate at 30 &#176;C for 12 hr.</li>
	<li>Scrape the colonies using a cell scraper and collect cells into a 50 ml conical tube using ice-cold cell storage media.</li>
	<li>Centrifuge at 1,000 x g for 20 min at 4 &#176;C. Resuspend the pellet in 10 ml ice-cold cell storage media to reach an OD600 of 100.</li>
	<li>Aliquot 20 &#181;l of the cells for storage at &#8722;70 &#176;C.</li>
</ol>
2. Removing False Positives from the Metagenomic Library
<ol>
	<li>Thaw the stock metagenomic library cells (from Step 1.13) containing metagenomic DNA fosmid and pGESS(E135K) on ice.</li>
	<li>Inoculate 10 &#181;l of the cells in 2 ml LB containing 50 &#181;g/ml ampicillin and 12.5 &#181;g/ml chloramphenicol in a 14-ml round-bottom tube. Incubate at 37 &#176;C with shaking at 200 rpm for 4 hr.</li>
	<li>Meanwhile, turn on the FACS machine and open the default FACS software. Use the following settings: nozzle tip diameter, 70 &#181;m; Forward Scatter Area (FSC-A) sensitivity, 300 V-logarithmic amplification; Side Scatter Area (SSC-A) sensitivity, 350 V-logarithmic amplification; Fluorescein Isothiocyanate Area (FITC-A) sensitivity, 450 V-logarithmic amplification; threshold parameter, FSC-A value 5. 
	Note: Typical settings are given for FACS device mentioned in the Table of Materials. Adjust the settings for other FACS devices.</li>
	<li>Dilute the metagenomic library cells from Step 2.2 by adding 5 &#181;l of the sample to a 5-ml round bottom tube containing 1 ml PBS.</li>
	<li>Place the diluted library sample onto the loading port of the FACS instrument and click on Load button on the Acquisition Dashboard of the FACS software.</li>
	<li>Adjust the event rate to 1,000 - 1,500 events/sec by clicking and controlling the Flow Rate buttons on the dashboard.</li>
	<li>Create log scaled FSC-A vs. log scaled SSC-A scatter plot on a global worksheet by clicking on the Dot Plot button in the tool bar. Adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button in the tool bar.</li>
	<li>Plot a histogram with cell count vs. log scaled FITC-A on the worksheet by clicking on Histogram button. Then, adjust the FITC voltage from the Cytometer Settings tab on the Inspector Control window such that the peak of the bell-shaped distribution is less than 102 of FITC-A.</li>
	<li>Create a log scaled FSC-A vs. the log FITC-A plot by clicking on the Dot Plot button on the global worksheet. Set a sorting gate R2 on the plot using the Polygon Gate button on the tool bar so that the gate is located between +5% and -5% cells from the center of the distribution (total 10% cells around the peak of the bell-shaped curve).</li>
	<li>Place a collection tube containing 1.2 ml LB containing 50 &#181;g/ml ampicillin and 12.5 &#181;g/ml chloramphenicol at the outlet of the FACS instrument and sort out 106 cells satisfying both the R1 and R2 gates.</li>
	<li>Remove the collection tube, cap, and gently vortex.</li>
</ol>
3. Metagenomic Enzyme Screening
<ol>
	<li>Incubate the sorted cells (from Step 2.11) at 37 &#176;C with shaking at 200 rpm until the OD600 reaches 0.5.</li>
	<li>Add 1 &#956;l copy induction solution to amplify the intracellular fosmid copy number. Incubate the cells for an additional 3 hr at 37 &#176;C with shaking at 250 rpm.</li>
	<li>To prepare cells for sorting, add 0.5 ml of the cultured cells and an appropriate substrate (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, or phenyl phosphate) into a 14-ml round-bottom tube at a final concentration of 100 &#181;M.
	<ol>
		<li>For a control sample, add 0.5 ml of the cultured cells from Step 3.2 into a 14-ml round-bottom tube. Add the same volume of PBS as the substrate volume in Step 3.3.</li>
	</ol>
	</li>
	<li>Incubate the two samples at 37 &#176;C with shaking at 200 rpm for 3 hr.</li>
	<li>Meanwhile, prepare the FACS machine with the same configurations as Step 2.3.</li>
	<li>Add 5 &#181;l of the cells (for sorting) and control cells to two 5 ml round-bottom tubes containing 1 ml PBS, respectively.</li>
	<li>Place the tube containing control cells onto the loading port of the FACS and click on Load button on Acquisition Dashboard of the FACS software. Adjust the event Rate to 1,000 - 3,000 events/sec by controlling Flow Rate buttons on the dashboard.</li>
	<li>Create log scaled FSC-A vs. log scaled SSC-A scatter plot by clicking on Dot Plot button on the global worksheet of the software and adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button on the tool bar.</li>
	<li>Create log scaled FSC-A vs. log scaled FITC-A scatter plot by clicking on the Dot Plot button on the worksheet and set a sorting gate R2 on the plot using the Polygon Gate button on the tool bar so that less than 0.1 % of negative cells are detected within the R2 gate.</li>
	<li>Replace the control sample tube with the sorting sample tube, and adjust the event rate to 1,000 - 3,000 events/sec by controlling the Flow Rate buttons on the dashboard.</li>
	<li>Place a collection tube containing 0.5 ml LB at the outlet of the FACS instrument and sort out 104 cells satisfying both the R1 and R2 gates. 
	Note: The sorting criterion can range from top 0.1% to 5% of FITC-A in the R2 gate. In this protocol, top 1% cells were collected as positives.</li>
	<li>Remove the collection tube, cap, and gently vortex.</li>
	<li>Spread the 0.5 ml collected samples on two agar plates (90 mm Petri dishes) containing LB, 50 &#181;g/ml ampicillin, 12.5 &#181;g/ml chloramphenicol and incubate the plate at 37 &#176;C overnight.</li>
	<li>If necessary, perform additional rounds of sorting for FITC-A enrichment by repeating Steps 3.1-3.14.</li>
</ol>
4. Hit Selection and Enzyme Activity Confirmation
<ol>
	<li>Flow cytometry analysis
	<ol>
		<li>From the incubated plate at Step 3.14, select a colony showing no fluorescence using a colony picker under the observation of 488 nm wavelength of a LED illuminator.</li>
		<li>Inoculate the selected colony in a 14-ml round-bottomed tube containing 2 ml LB containing 50 &#181;g/ml ampicillin and 12.5 &#181;g/ml chloramphenicol at 37 &#176;C with shaking at 200 rpm until its OD600 reaches 0.5.</li>
		<li>Add 2 &#956;l copy induction solution to amplify the intracellular fosmid copy number. Incubate the cells for an additional 3 hr at 37 &#176;C with shaking at 250 rpm.</li>
		<li>Prepare a 14 ml round-bottom tube and add 1 ml of the cultured cells (Step 4.1.3). Add an appropriate substrate (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, or phenyl phosphate) at a final concentration of 100 &#181;M.
		<ol>
			<li>For a control, add the 1 ml cultured cells from Step 4.1.3 into a 14 ml round-bottom tube and add the same volume of PBS as the substrate volume in Step 4.1.4.</li>
		</ol>
		</li>
		<li>Incubate the two samples at 37 &#176;C with shaking at 200 rpm for 3 hr.</li>
		<li>Meanwhile, prepare the FACS machine with the same configurations as Step 2.3.</li>
		<li>Add 5 &#181;l of the control and substrate treated cells to 5 ml round-bottom tubes containing 1 ml PBS, respectively.</li>
		<li>Place the tube containing control cells on the loading port of the FACS device and click on Load button on Acquisition Dashboard of the FACS software. Adjust the event rate to 1,000 - 3,000 events/sec using Flow Rate buttons on the dashboard.</li>
		<li>Click on Acquisition button to measure FITC-A.</li>
		<li>Replace the control sample tube with the substrate treated sample tube. Adjust the event rate to 1,000 - 3,000 events/sec using Flow Rate buttons on the dashboard.</li>
		<li>Click on Acquisition button to measure FITC-A.</li>
		<li>Compare the fluorescence of the two groups of cells by plotting a histogram of cell count vs. the log scaled FITC-A.</li>
	</ol>
	</li>
	<li>Other assays to confirm enzyme activity
	<ol>
		<li>For further identification of the selected enzyme candidates, extract fosmid DNA using standard extraction procedures from the commercial extraction kits and analyze the nucleotide sequence, or test the in vitro enzyme activity6,7.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Increasing production efficiency of biocatalysts is a key for the success of bio-chemical based industry9 and metagenome is considered one of the best natural enzyme source. In this sense, it is essential to screening novel enzymes from the metagenome where majority of the genetic resources have not been explored10. Several screening methods have been developed which directly detect enzyme products using transcriptional activators11, 12 but these techniques require specific metabolite-res...

A recently developed high-throughput single-cell assay technique allows novel enzymes to be screened from a large-scale genetic library based on their functional activities1. At the single cell level, proteins regulating transcription are employed to trigger reporter gene expression by sensing small molecules that are produced as a result of a target enzyme activity. One early approach involved the isolation of a phenol-degrading operon from Ralstonia eutropha E2 using the substrate-induced genetic expression screening (SIGEX) method, in which the substrate induces the expression of a reporter protein2. NhaR of Pseudomonas putida was used to select benzaldehyde dehydrogenase3, and LysG from Corynebacterium glutamicum was utilized for the high-throughput screening of a new L-lysine-producing strain from diverse mutant libraries4.
Previously, a genetic enzyme screening system (GESS) was proposed as a generally applicable screening platform5. This system uses the phenol-recognizing dimethylphenol regulator, DmpR, of P. putida. DmpR(E135K), and a mutant of DmpR, can also be employed in GESS (pNP-GESS) for the detection of p-nitrophenol (pNP). In the presence of target enzymes producing phenolic compounds, GESS in E. coli cells emits a fluorescence signal, enabling the rapid isolation of single cells using a fluorescence-activated cell sorter (FACS). But the expression of metagenomic enzyme appears to be weaker than that of conventional recombinant enzymes; therefore, GESS was designed to detect phenolic compounds with maximum sensitivity by investigating the combination of ribosomal binding site (RBS) and terminator sequences along with optimal operating condition5.
One of the fundamental advantages of GESS is that this single method theoretically allows the screening of over than 200 different types of enzymes in the BRENDA database (Table 1, http:// www.brenda-enzymes.info, 2013.7) by simply employing different substrates. It was shown that cellulase, lipase, and methyl parathion hydrolase (MPH) can be detected using pNP-GESS with appropriate substrates of p-nitrophenyl butyrate, p-nitrophenyl-cellotrioside, and methyl parathion, respectively5. Recently, it was proved that an alkaline phosphatase (AP), which is one of the novel enzymes identified using pNP-GESS, is the first thermolabile AP found in cold-adapted marine metagenomes6.
Here, details of the screening process is presented with pNP-GESS detecting the activities of three different types of enzymes- lipase, cellulase, and alkaline phosphatase -and rapidly identifying novel candidate enzymes from a metagenomic library5,6. The processes include metagenome preprocessing with pNP-GESS and operating a flow cytometry sorter. While the hits obtained will need to be sequenced for further identification, this protocol covers the procedure up to the steps of enzyme activity confirmation using flow cytometry.

<table><tbody><tr><td>CopyControl</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid library production kit&amp;nbsp;</td></tr><tr><td>CopyControl Induction Solution</td><td>Epicentre</td><td>CCIS125</td><td>Fosmid copy induction solution</td></tr><tr><td>EPI300</td><td>Epicentre</td><td>EC300110</td><td>Electrocompetent cell</td></tr><tr><td>pCC1FOS</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid vector</td></tr><tr><td>Gene Pulser Mxcell</td><td>Bio-Rad</td><td></td><td>Electroporation cuvette and electroporate system</td></tr><tr><td>FACSAria III</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry (FACS machine)</td></tr><tr><td>AZ100M</td><td>Nikon</td><td></td><td>Microscope&amp;nbsp;</td></tr><tr><td>UltraSlim&amp;nbsp;</td><td>Maestrogen</td><td></td><td>LED illuminator</td></tr><tr><td>50-ml conical tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>14-ml round-bottom tube&amp;nbsp;</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>5-ml round-bottom tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma-Aldrich</td><td>N7653</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl &amp;beta;-D-cellobioside</td><td>Sigma-Aldrich</td><td>N5759</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butylate</td><td>Sigma-Aldrich</td><td>N9876&amp;nbsp;</td><td>Substrate</td></tr><tr><td>Luria-Bertani (LB)</td><td>BD Difco</td><td>244620</td><td>Tryptone 10 g/L, Yeast extract 5 g/L, Sodium Chloride 10 g/L</td></tr><tr><td>Super Optimal broth (SOB)</td><td>BD Difco</td><td>244310</td><td>Tryptone 20 g/L, Yeast extract 5 g/L, Sodium Chloride 0.5 g/L, Magnesium Sulfate 2.4 g/L, Potassium Chloride 186 mg/L</td></tr><tr><td>Super Optimal broth with Catabolite repression (SOC)</td><td></td><td>SOB, 0.4 % glucose</td><td></td></tr><tr><td>2x Yeast Extract Tryptone (2xYT)</td><td>BD Difco</td><td>244020</td><td>Pancreatic digest of Casein 16 g/L, Yeast extract 10 g/L, Yeast extract 5 g/L</td></tr><tr><td>Cell storage media</td><td></td><td></td><td>2x YT broth, 15% Glycerol, 2% Glucose</td></tr><tr><td>pGESS(E135K)</td><td></td><td></td><td>A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.&lt;br/&gt; See the reference 5 in the manuscript for more details.</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry Software Version 7.0</td></tr><tr><td>PBS</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr></tbody></table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

The three phenolic substrates were examined to identify novel metagenomic enzymes from a metagenome library of ocean-tidal flat sediments in Taean, South Korea by following the proposed protocol. For the library construction, average 30 - 40 kb metagenome sequences were inserted into fosmids, which are based on the E. coli F factor replicon and presented as a single copy in a cell. Note that fosmids have been widely used for constructing complex genomic libraries due to their sta...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

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Biology

8.7K Views.  Korea Research Institute of Bioscience and Biotechnology. This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate). 

Video: Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biochemistry

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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

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

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

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

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

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

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

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

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be &#8220;forward&#8221;, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be &#8220;reverse&#8221;, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic screens, including those that depend on either loss-of-function or gain-of-function mutations, which respectively decrease or increase the activity of genes. We then explore general protocols for forward and reverse screens in a popular model organism, the nematode worm. Finally, we highlight how screens are applied in research today, for example to better understand gene interactions that may contribute to neurodegenerative diseases.

Forward and Reverse Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

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High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

High-throughput Yeast Plasmid Overexpression Screen

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

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

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

Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

Genetic Screens