We thank John Darga of the MGH DNA Core for preparation of the DNA screening library. Christopher Chigas of Perkin Elmer provided invaluable support for our Wallac 1420 luminometer. Steven Ciacco and Martin Thomae of Agilent provided support for the Bravo robot. We thank Ron Johnson and Steve Titus at the NIH for generously providing their dual-glow luciferase assay protocol.

For an experimental overview, see Figure 1.
1. Prepare Reporter Plasmids Containing the Alpha-Synuclein Promoter
Regulatory elements of the alpha-synuclein gene (Figure 2) span approximately 10 kb, from the upstream NACP (non-A beta component of amyloid peptide) dinucleotide repeat sequence1,2 through intron 2 3. We include a portion of this region in our reporter construct. We found that plasmids containing intron 2 required special procedures for growth, as outlined below. The luciferase plasmids, pGL4.10 and pGL4.75 (Figure 3), are commercially available from Promega and can be propagated using conventional molecular biology protocols.
<ol>
	<li>Amplify the 2&#160;components of the alpha-synuclein promoter in separate PCRs using the recipe in Table 1 and the primers in Table 2.</li>
	<li>Verify PCR products on an agarose gel and clean using the Qiaquick PCR Purification kit (Qiagen), eluting in 50 &#956;l of TE (Tris-EDTA). Store the eluted 0.9 kb fragment at -20 &#176;C for future use.</li>
	<li>Digest the entire volume of the 3.4 kb PCR fragment, as well as 5 &#956;g of pGL4.10, with SacI and XhoI. Treat the vector with Calf Intestine Alkaline Phosphatase (Roche) and gel purify using the PureLink Quick Gel Extraction kit (Invitrogen).</li>
	<li>Ligate the fragment and vector using T4 DNA ligase (NEB). Clean the completed reaction using the PureLink PCR Micro kit (Invitrogen), eluting in 10 &#956;l TE buffer.</li>
	<li>Transform 2 &#956;l of the cleaned, ligation reaction into 20 &#956;l of ElectroMAX Stbl4 competent cells (Invitrogen) and electroporate according to the manufacturer&#39;s instructions. Shake in a 30 &#176;C incubator at 225 rpm for 90 min. Spread 2&#160;volumes on LB plates containing 100 mg/ml ampicillin and incubate at 30 &#176;C for 2&#160;days.</li>
	<li>Using a toothpick, select 4-6 small colonies for inoculation into 2 ml of TB (Terrific Broth). Grow these miniprep cultures in a 30 &#176;C shaker O/N.</li>
	<li>Purify plasmid DNA from 1.5 ml of each miniprep culture using the PureLink HiPure Plasmid Miniprep kit (Invitrogen).</li>
	<li>Digest the minipreps with BamHI and electrophorese on an agarose gel. The correct clone should produce fragments of 2.8 kb and 4.9 kb.</li>
	<li>Restreak the remaining miniprep culture from the correct clone on a fresh LB (Luria Broth) plate and grow at 30 &#176;C for 2&#160;days.</li>
	<li>Select a small colony and inoculate a 1.5 ml TB starter culture. Shake O/N at 30 &#176;C. Do not let the starter culture grow to saturation.</li>
	<li>Dilute the entire starter culture into 150 ml of prewarmed TB and shake at 30 &#176;C for 2&#160;days. Before proceeding to the next step, use 1.5 ml of this culture for an additional miniprep and digestion to confirm that the clone did not mutate during replication.</li>
	<li>Purify plasmid DNA from the remaining culture using the PureLink HiPure Plasmid Maxiprep kit (Invitrogen). Expected yields of this intermediate plasmid are 50-150 &#956;g per 150 ml of culture.</li>
	<li>Thaw the 0.9 kb fragment frozen in Step 1.2. Repeat Steps 1.3 to 1.12 to clone the 0.9 kb fragment into the intermediate plasmid, digesting instead with KpnI and SacI. Ligate, transform, select, and grow for maxipreps as above. Digestion with BamHI should yield fragments of 5.8 kb and 2.8 kb. This is the plasmid that will be used for the screen.</li>
</ol>
2. Prepare 293T Cells, Reporter Plasmids, and Library DNA for Screening
293T cells are first seeded into 96-well plates and transfected the following day. Reporter plasmids and library DNAs are diluted into 96-well plates. We obtained plates of transfection-grade library DNA from the DNA Core at Massachusetts General Hospital (<a href="http://dnacore.mgh.harvard.edu" target="_blank">dnacore.mgh.harvard.edu</a>). This protocol transfects a single library plate into 2&#160;identical plates of 293T cells (Figure 1), thus 2&#160;plates of cells should be seeded for each library plate to be screened.
Note: Grow cells in media without phenol red or antibiotics, as these interfere with the luciferase assays and increase toxicity during transfection, respectively.
<ol>
	<li>For each library plate to be screened, resuspend trypsinized 293T cells to a concentration of 1.5 x 105 cells/ml in DMEM containing 14% FBS. Pour into a sterile reservoir (Axygen).</li>
	<li>Place the reservoir, 2&#160;clear-bottomed, 96-well plates (Corning), and a box of filter pipet tips (Agilent) on the robot deck. Dispense 100 &#956;l of cells into each well of both plates, for a density of 1.5 x 104 cells per well.</li>
	<li>Centrifuge the cell plates at 50 x&#160;g for 2 min, using low ramp speeds to ensure equal cell density throughout each well. Incubate at 37 &#176;C and 10% CO2 O/N.</li>
	<li>Dilute the firefly and Renilla reporter plasmids to 5 ng/&#956;l in serum-free media. Combine the dilutions in a ratio of 5:3 firefly to Renilla plasmid (by volume) in a 15 ml conical tube.</li>
	<li>Pipet the combined plasmids into each well of a 96-well plate, dispensing 17 &#956;l per library plate, plus 2-10 &#956;l excess, into each well.</li>
	<li>Obtain transfection-grade DNA library plates as above and dilute to 13.3 ng/&#956;l with endotoxin-free water. The minimum volume per well should be 28 &#956;l.</li>
</ol>
3. Perform Transfections
On day 2&#160;of the screen, reporter plasmids and library DNAs are transfected into 293T cells.
<ol>
	<li>For each library plate, mix 107.5 &#956;l of RT Optifect (Invitrogen) with 4.3 ml serum-free media (1:40 dilution) in a polystyrene tube. Incubate for 5 min at RT, and then dispense 44 &#956;l (per library plate) into each well of a 96-well, polystyrene V-bottomed plate (Greiner).</li>
	<li>Place the plate of diluted Optifect, reporter plasmids (Step 2.5), library DNAs (Step 2.6), and a box of pipet tips on the robot deck.</li>
	<li>Aspirate 17 &#956;l of reporter plasmids, 43 &#956;l of diluted Optifect, and 26 &#956;l of library DNA, inserting a 5 &#956;l air gap between each aspiration. The library DNA should be aspirated last to prevent cross-contamination. Dispense the contents of the tips into a new polystyrene V-bottomed plate, mix, cover, and set aside for 20 min to allow lipid/DNA complexes to form. If transfecting multiple library plates, replace the pipet tips and library plate, and repeat.</li>
	<li>Uncover the Optifect/DNA mixture plate and place it on the robot deck with 2 plates of 293T cells. Using a single box of pipet tips, aspirate 80 &#956;l of the Optifect:DNA mixture and slowly dispense 40 &#956;l onto each plate of cells. If transfecting multiple library plates, repeat for all Optifect:DNA plates. Cover cells.</li>
	<li>Centrifuge the cell plates at 1,200 x&#160;g for 30 min. Cover and return to the incubator.</li>
	<li>Incubate the cells for 4-6 hr. During this incubation, prepare fresh media by mixing 12 ml DMEM containing 10% FBS and 10 &#956;g/ml ciprofloxacin (CellGro) for each transfected library plate. Pour fresh media into a sterile reservoir.</li>
	<li>Place 2&#160;boxes of robot tips, a single plate of cells, the reservoir containing fresh media, and a waste reservoir onto the robot deck. Aspirate 100 &#956;l of media from the cells and dispense into the waste reservoir partially filled with 70% ethanol. Using fresh tips, aspirate 100 &#956;l of fresh media and slowly dispense onto the cells. Repeat for all plates of cells.</li>
	<li>Return plates to the incubator for 48 hr.</li>
</ol>
4. Perform Dual-Luciferase Assays
On day 4&#160;of the screen, the firefly and Renilla luciferase assays are performed.
Note: The dual-luciferase assay requires the sequential addition of 2&#160;assay buffers, 3X firefly assay buffer and 3X Renilla assay buffer, as described in Table 3. Firefly and Renilla luciferases are not secreted, thus cells must be lysed prior to measuring luciferase activity. Firefly assay buffer lyses cells and provides substrate for the firefly luciferase; Renilla assay buffer quenches the firefly signal and provides substrate for the Renilla luciferase.
<ol>
	<li>For each library plate, prepare 8 ml of 3X firefly assay buffer from stock solutions as described in Table 3, and dispense 82 &#956;l into each well of a 96-well V-bottomed plate.</li>
	<li>For each library plate, also prepare 12 ml of 3X Renilla assay buffer and dispense 122 &#956;l into each well of a 96-well V-bottomed plate. Set aside both the firefly and Renilla assay buffers.</li>
	<li>Place a box of pipet tips, a waste reservoir, and 2&#160;plates of cells (transfected from the same library plate) on the robot deck.</li>
	<li>Aspirate 60 &#956;l of media from each plate and discard in the waste reservoir partially filled with 70% ethanol. Cover plates and set aside. If transfecting multiple library plates, repeat for all sets of plates.</li>
	<li>Place 2&#160;boxes of pipet tips, the plate of 3X firefly assay buffer, and a set of cell plates on the robot deck.</li>
	<li>Aspirate 40 &#956;l of 3X firefly assay buffer and add it to the first plate of cells, mixing thoroughly. Switching to new pipet tips, repeat for the second cell plate. Place cells at RT for 10 min to complete lysis. If transfecting multiple library plates, replace the boxes of tips and cell plates, and repeat.</li>
	<li>Record luminescence from each well of each cell plate on a luminometer, recording for 1 sec per well. Return plates to the robot deck.</li>
	<li>Place 2&#160;boxes of pipet tips, the plate of 3X Renilla assay buffer, and a set of cell plates on the robot deck.</li>
	<li>Aspirate 60 &#956;l of 3X Renilla assay buffer, and add it to the first plate of cells, mixing thoroughly. Switching to a new box of pipet tips, repeat for the second cell plate. If transfecting multiple library plates, repeat for all sets of cell plates.</li>
	<li>Record luminescence from all plates on a luminometer, recording each well for 1 sec as above. Discard plates.</li>
</ol>
5. Data Analysis
<ol>
	<li>Calculate the firefly:Renilla luminescence ratio (&#34;F:R ratio&#34;) for each well by dividing the counts of firefly signal by the counts of Renilla signal.</li>
	<li>Calculate the induction value by dividing the F:R ratio of each well by the average F:R ratio of the plate, excluding the highest and lowest 25% of wells.</li>
	<li>Average the induction values across replicates for a single transfected library plate. Genes inducing or repressing alpha-synuclein more than three-folds are considered &#34;hits&#34; in this screen and are subject to further validation and secondary screens.</li>
</ol>

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This work was supported by royalties obtained by licensing BacMamreagents (US patent 5,731,182).

Alpha-synuclein has been implicated in Parkinson&#39;s disease (PD) as a component of Lewy bodies4, intracellular inclusions considered pathognomonic for the disease. Numerous genome-wide association studies have linked single nucleotide polymorphisms in alpha-synuclein with increased risk for sporadic PD5,6,7. Although less common than sporadic PD, familial PD may be also caused by mutations in alpha-synuclein8, as well as duplication and triplication of the alpha-synuclein locus9,1...

The ability to identify key genetic regulatory elements and the factors that act on them is fundamental for the exploration of numerous biological processes. However, identifying factors that regulate expression of genes in rare cell types, such as specific neuronal populations, can be challenging. Here, we present a protocol for identifying novel transcriptional regulators of alpha-synuclein (SNCA), a gene associated with Parkinson&#39;s disease and expressed in dopaminergic neurons in the substantianigra pars compacta region of the midbrain. We accomplish this using a high-throughput, dual-luciferase, in vitro reporter screen to deconstruct alpha-synuclein expression in 293T cells. The alpha-synuclein promoter is first cloned into a reporter plasmid containing the firefly luciferase gene. A commercially available plasmid containing the Renilla luciferase under the control of a constitutively active promoter serves as an internal control. These reporter constructs are co-transfected into 293T cells in microplates with plasmids from a DNA expression library, such that each well is transfected with a single library plasmid. After 48 hr, luciferase activity for each reporter is measured sequentially using a dual-glow assay. The relative expression of alpha-synuclein in response to each library gene is inferred by the ratio of firefly:Renilla luciferase activity in each well (F:R ratio) after plate-based normalization.
This protocol provides the ability to screen a large number of genes (~2,500 per week) for their ability to transactivate a reporter gene using minimal manpower (1-2 people) and minimal cost (about $3 reagent cost per microplate assay). Transcriptional regulators of neuronal genes (e.g., alpha-synuclein), which are difficult to study in cultured neurons refractory to genetic manipulation, can be compared side-by-side in this direct functional assay. We include in our protocol a detailed method for cloning and growth of plasmids containing the alpha-synuclein promoter, since we found that plasmids containing this region are unstable when grown with conventional procedures (see Discussion). We also include an inexpensive alternative to commercially available dual-luciferase reagents for high-throughput assays. This protocol can be easily adapted to target other regulatory elements of interest, or to any process requiring high-throughput transient transfections.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Typical luciferase values, F:R ratios and induction values for a single half-plate are shown in Figure 4. Note the hit in well H2, a 32-fold inducer. Genes causing excessive toxicity (for example, well E3), or wells that were poorly transfected, will produce low values for both firefly and Renilla luciferases, but an average induction value. Genes causing non-specific induction of luciferase activity, perhaps via interaction with the pGL4 backbone, will induce firefly and Renilla</e...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

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

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-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.4K Görüntüleme.  Massachusetts General Hospital.  Bu yüksek verimli robot transfections ve bir ev yapımı çift ışıma lusiferaz deneyi kullanılarak transkripsiyon düzenleyiciler belirlenmesi için hızlı ve ucuz bir tarama yöntemi sunulmaktadır. Bu protokol, hızlı bir şekilde binlerce gen için doğrudan yan yana fonksiyonel veriler üreten ve ilgi duyulan herhangi bir geni hedef kolayca değiştirilebilir olduğunu. 

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

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

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 ...