Name: colorimetric detection of bacteria using litmus test
Uploaded: 2016-09-17T13:00:00
Description: Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

1. Preparation of Reagents and Buffers
<ol>
	<li>0.5 M Ethylenediaminetetraacetic Acid (EDTA)
	<ol>
		<li>In a 2 L beaker, add 186.1 g EDTA to 800 ml of deionized-distilled water (ddH2O). Adjust the pH of the solution to 8.0 using NaOH pellets. Add ddH2O to a final volume of 1.0 L and transfer the solution to an autoclavable glass bottle for autoclaving and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>10&#215; Tris-borate EDTA (10x TBE)
	<ol>
		<li>In a 4 L plastic beaker, add 432 g Tris-base, 200 ...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=70+Years+of+crystalline+urease:+What+have+we+learned.">70 Years of crystalline urease: What have we learned.</a> Acc. Chem. Res. 30, 330-337 (1997).</li><li>Omiccioli, E., Amagliani, G., Brandi, G., Magnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+platform+for+Real-Time+PCR+detection+of+Salmonella+spp.,+Listeria+monocytogenes+and+Escherichia+coli+O157+in+milk.">A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk.</a> Food Microbiol. 26, 615-622 (2009).</li><li>Cui, S., Schroeder, C. M., Zhang, D. 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The translation of the action of the RNA cleavage activity of a bacterium-responsive DNAzyme to a litmus test is made possible through the use of urease and magnetic separation, as illustrated by Figure 1. Although the demonstration of the modified litmus test for bacterial detection is done with an E. coli-dependent RNA-cleaving DNAzyme,5,19,20 the design can be generally extended for any RNA-cleaving DNAzyme. Given the great availability of RNA-cleaving DNAzymes for different analyt...

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:183px;">VWR AMRESCO</td>
			<td style="width:163px;">0105</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide (NaOH) pellets</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>SB6789</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tris-base</td>
			<td>VWR AMRESCO</td>
			<td>0497</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Boric acid</td>
			<td>AMRESCO</td>
			<td>0588</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urea</td>
			<td>VWR AMRESCO</td>
			<td>M123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">40% acrylamide/bisacrylamide (29:1) solution</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>A0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sucrose</td>
			<td>Bioshop Canada inc.</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Bromophenol blue</td>
			<td>Bioshop Canada inc.</td>
			<td>BRO777</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Xylenecyanol FF</td>
			<td>SIGMA-ALDRICH</td>
			<td>X-4126</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% sodium dodecyl sulfate</td>
			<td>Bioshop Canada inc.</td>
			<td>SDS001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hydrochloric Acid (HCl)</td>
			<td>CALEDON LABORATORIES LTD</td>
			<td>6026</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Chloride (NaCl)</td>
			<td>Bioshop Canada inc.</td>
			<td>SOD001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Bioshop Canada inc.</td>
			<td>HEP001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnesium Chloride (II) hexahydrate</td>
			<td>VWR AMRESCO</td>
			<td>0288</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tween 20</td>
			<td>Bioshop Canada inc.</td>
			<td>TW508</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Adenosine Triphospahte (ATP)</td>
			<td>AMRESCO</td>
			<td>0220</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Acetate trihydrate (NaOAc)</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tetramethyleneethylenediamine (TEMED)</td>
			<td>AMRESCO</td>
			<td>0761</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% Ammonium persulfate (APS)</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>AB0072</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>22360</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl sulfoxide (DMSO)</td>
			<td>CALEDON LABORATORIES</td>
			<td>803540</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urease</td>
			<td>SIGMA-ALDRICH</td>
			<td>U0251</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1&#215; Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>70011-069</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.04% Phenol red</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215;T4 polynucleotide kinase reaction buffer</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215; T4 DNA ligase reaction buffer</td>
			<td>Bio Basics Canada</td>
			<td>B1122-B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 DNA ligase (5 U/uL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Luria Bertani (LB) Broth</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 polynucleotide kinase (10 U/uL)</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E. coli K12 (MG1655)</td>
			<td>ATCC</td>
			<td>ATCC700926</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Centrifuge</td>
			<td>Beckman Coulter, Inc.</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Glass plates</td>
			<td>CBS scientific</td>
			<td>ngp-250nr</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.75 mm thick spacers</td>
			<td>CBS scientific</td>
			<td>VGS-0725r</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-well comb</td>
			<td>CBS scientific</td>
			<td>VGC-7512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">UV Lamp</td>
			<td>UVP</td>
			<td>95-0017-09</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Spectrophotometer (NanoVue)</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Metal plate</td>
			<td>CBS scientific</td>
			<td>CPA165-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vortex</td>
			<td>VWR International</td>
			<td>58816-123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gel electrophoresis apparatus</td>
			<td>CBS scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Petri dishes</td>
			<td>VWR International</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">100 kDa MWCO centrifugal filters</td>
			<td>EMD Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Bead (BioMag)</td>
			<td>Bangs Laboratories Inc</td>
			<td>BM568</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Seperation Rack</td>
			<td>New England BioLabs</td>
			<td>S1506S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72.69</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Syringe filter (0.22 um)</td>
			<td>VWR International</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14 mL culture tube</td>
			<td>VWR International</td>
			<td>60818-725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture incubator</td>
			<td>Eppendorf Scientific</td>
			<td>M13520000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Branson Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Camera (Canon Powershot G11)</td>
			<td>Canon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL conical tube</td>
			<td>VWR International</td>
			<td>89004-364</td>
			<td></td>
		</tr>
	</tbody>
</table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

The principle of the bacterial litmus test is explained in Figure 1. The test uses three key materials: an RNA-cleaving DNAzyme that is activated by a specific bacterium, urease and magnetic beads. The DNAzyme is used as the molecular recognition element to achieve highly specific detection of a bacterium of interest. Urease and magnetic beads are used to achieve signal transduction of the RNA-cleavage activity of the DNAzyme. This involves the creation of magnetic beads ...

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

The overall goal of this experiment is to demonstrate a simple modified litmus test for bacterial detection. This method describes how an E.coli-activated RNA-cleaving DNA enzyme can be coupled with a urease-based signal trandsduction platform to generate a color change in response to E.coli. The two main advantages of this technique are the simplicity of the test and its applicability to other analyte-responsive RNA-cleaving DNA enzymes.Demonstrating the procedure will be Seperh Manchehry and Dingran Chang, graduate students from Dr.Yingfu Li's lab at McMaster University. After preparing reagents and buffers according to the text protocol, prepare a stock solution of succinimidyl 4-cyclohexane-1-carboxylate, or SMCC, by dissolving one milligram of SMCC in 676 microliters of DMSO. Vortex and place on ice until use.Dissolve one milligram of urease in milliliter of 1x PBS and also place that on ice until use. To synthesize urease DNA, or UrDNA, add 10 microliters of 100 micromolar LD1 to a 2.5 milliliter microfuge tube. Then, add 140 microliters of doubly-distilled water and 40 microliters of 10x PBS and vortex.Next, add 80.5 microliters of SMCC stock and 159.5 microliters of DMSO. Then, vortex again and use a benchtop centrifuge to briefly spin. Incubate the reaction at 37 degrees Celsius in an incubator or a heat block for 60 minutes.Following the incubation, add 200 microliters of 1x PBS, 60 microliters of three-molar sodium acetate, and 1.5 milliliters of cold 100%ethanol to the tube. Mix by vortexing, and incubate the reaction at minus 20 degrees Celsius for 30 minutes. Centrifuge the solution at 20, 000 times g and four degrees Celsius for 20 minutes.Then, remove the supernatant and dry the pellet under vacuum. To the dried pellet, add 400 microliters of urease stock and incubate at room temperature for 5 hours. Transfer 200 microliters of crude conjugate to a pre-washed 100, 000 molecular weight cutoff centrifugal filter column.Centrifuge the column at 14, 000 times g for five minutes. Transfer the remaining 200 microliters of crude conjugate to the column and centrifuge at 14, 000 times g for five minutes. Then, remove the column and place it upside down in a new two-milliliter microfuge collection tube.Centrifuge the inverted column and tube at 1, 000 times g for two minutes. After removing the collection tube, add 30 microliters of 1x PBS to the centrifugal column to wash the membrane for additional recovery of conjugates. Invert the column again and place it back into the collection tube.Centrifuge the column at 1, 000 times g for two minutes. Remove and dispose of the column. Store the UrDNA at four degrees Celsius until use.To assemble an E.coli-activated RNA-cleaving DNAzyme, or EC1, and UrDNA onto magnetic beads, mix the magnetic bead stock well and transfer 100 microliters of bead suspension to a 1.5 milliliter microfuge tube. Then, place the tube on a magnetic rack holder for isolating the beads. Remove the supernatant by pipetting and add 150 microliters of binding buffer, or BB, to the tube.Remove the tube from the holder and carefully tap the tube to re-suspend the beads into a homogeneous solution. After repeating the BB wash two more times with the beads in suspension with BB, add 10 microliters of 10 micromolar EC1. Carefully mix by tapping on the tube.Incubate the solution with mild shaking for 30 minutes. To avoid aggregation of the beads, tap on the tube every two to three minutes. Next, place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.Then, use 150 microliters of BB to wash the beads three times. Once the washing is complete, suspend the beads in a total of 150 microliters of BB.To this solution, add 15 microliters of UrDNA and heat the sample at 45 degrees Celsius for two minutes, then cool the solution to room temperature and incubate for two hours. Place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.Add 100 microliters of reaction buffer, or RB, then remove the tube from the magnetic rack and carefully re-suspend the beads. After preparing E.coli cells according to the text protocol, pre-wash a 1.5 milliliter microfuge tube by adding and vortexing 100 microliters of RB.Then, discard the buffer. Add 15 microliters of assembled EC1 to the washed tube, then place the tube on the magnetic rack and aspirate the supernatant.Remove the tube from the rack, add 100 microliters of RB, and carefully re-suspend the magnetic beads. After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E.coli sample to the tube and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.After the incubation, add 90 microliters of doubly-distilled water and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5 milliliter microfuge tube. It is very important to pipette the solution carefully without taking up any of the magnetic beads.These beads are loaded with urease and will generate a false positive signal. Add 15 microliters of 0.04%Phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.The starting color of the litmus test must be yellow, reflecting a solution pH below 5.5. If necessary, the starting pH of the litmus test can be adjusted with a weak buffer, such as one millimolar sodium acetate buffer at pH five. This figure shows the results of the bacterial litmus test, where E.coli-activated RNA-cleaving DNAzyme EC1 was used as the DNAzyme and Phenol red was used as the pH indicator.As shown by the lack of color change in the absence of E.coli, EC1 is highly specific and exhibits minimal activity towards other bacteria. The depth of color change is dependent both on the number of E.coli cells used in the DNAzyme activation step and on the time allowed for the urea hydrolysis step. More E.coli cells result in a stronger color change, and a longer time for urea hydrolysis allows for the detection of a smaller number of cells.This graph shows the pH change as monitored using a handheld pH meter. The presence of 10 to the seven E.coli cells resulted in a gradual increase in pH by three units within 10 minutes. In contrast, the absence of E.coli did not cause detectable pH changes in the same conditions.Once mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it is important to remember that urease is very active, and poor washing or contamination will lead to a false positive. Following this procedure, this method can be performed to test for E.coli in more relevant conditions, such as testing for environmental water quality.After watching this video, you should have a good understanding for taking advantage of a simple litmus test for E.coli testing. Don't forget that this test can be used to detect pathogenic E.coli, which is extremely hazardous. You need to take biosafety training before conducting any of the tests for pathogenic bacteria.

colorimetric-detection-of-bacteria-using-litmus-test

Research

JoVE Journal

Biology

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

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.

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 ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.