|
|
| (8 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | {{Team Toronto Page Mainmenu bar}}
| |
| - | {{Team toronto page CSS assays}}
| |
| | | | |
| - | <html>
| |
| - | <head>
| |
| - | <link href='http://fonts.googleapis.com/css?family=Open+Sans+Condensed:300' rel='stylesheet' type='text/css'>
| |
| - | <style>
| |
| - |
| |
| - |
| |
| - | div.scroll
| |
| - | {
| |
| - | position: relative;
| |
| - | width:810px;
| |
| - | height:950px;
| |
| - | border-color: #A7A7A7;
| |
| - | overflow:scroll;
| |
| - | z-index: 250;
| |
| - | left:145px;
| |
| - | top:560px;
| |
| - | background-color:#FFFFCC;
| |
| - | opacity:0.7;
| |
| - |
| |
| - | }
| |
| - |
| |
| - |
| |
| - | }
| |
| - | #search-controls{/* la barre de recherche*/
| |
| - | display:none;
| |
| - | }
| |
| - | div.scroll p
| |
| - | {
| |
| - | margin:30px 50px;
| |
| - | }
| |
| - |
| |
| - | div.ex
| |
| - | {
| |
| - | z-index:500;
| |
| - | }
| |
| - | </style>
| |
| - |
| |
| - | </head>
| |
| - |
| |
| - | <body>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="scroll">
| |
| - | <h7><font color=black><p style = "text-align:center; font-size:35px;"><b>Assay Protocols</b></p><br/>
| |
| - |
| |
| - | <p style = "font-size:18px;"><b><u>Cell density</u></b><br/>
| |
| - | <b>Principle:</b> OD<sup>600</sup> is used as a surrogate for number of cells.
| |
| - | <br><b>Materials:</b>
| |
| - | <br> •Plate reader
| |
| - | <br> •96-well plate with clear bottom
| |
| - | <br>
| |
| - | <b>Procedure:</b>
| |
| - | <br> 1. Measure OD<sup>600</sup> for the entire plate of cultures in the micrometer plate
| |
| - | reader.
| |
| - | <br>
| |
| - |
| |
| - | <br><b><u>Aggregation</u></b><br/>
| |
| - | <b>Principle:</b> Measure cell density in culture below surface, aggregated cells will have
| |
| - | sunk to the bottom of the well.
| |
| - | <br>
| |
| - | <b>Materials</b>
| |
| - | <br> •Plate reader
| |
| - | <br> •96-well plate with clear bottom
| |
| - | <br>
| |
| - | <b>Procedure:</b>
| |
| - | <br> 1. Remove 150 μL of culture volume from overnight culture plates.
| |
| - | <br> 2. Place it into the empty microtiter well, at the edge of the plate. To prevent
| |
| - | bubble formation, make sure to stop pipetting at the first stop.
| |
| - | <br> 3. Measure OD<sup>600</sup><br>
| |
| - |
| |
| - | <br><b><u>Crystal violet</u></b><br/>
| |
| - | <b>Principle:</b> Crystal violet binds to polysaccharides of the biofilm matrix. By measuring
| |
| - | the binding to adhering biofilm, the amount of biofilm produced can be quantified. A
| |
| - | stock solution of crystal violet is incubated with the culture, the supernatant is
| |
| - | removed, bound crystal violet is mobilised with an ethanol:acetone solution and the
| |
| - | absorption is determined.<br>
| |
| - | <b>Materials:</b><br>
| |
| - | •Crystal Violet stock solution: To make 1L, add 0.3 g of crystal violet to a bottle
| |
| - | and fill to 1L with double distilled water. Stir well. Store at room temperature
| |
| - | till usage.<br>
| |
| - | •1400 μl double distilled water<br>
| |
| - | •350 μL 80:20 ethanol:acetone stock solution<br>
| |
| - | •96-well plate with clear bottom<br>
| |
| - | <b>Procedure:</b><br>
| |
| - | 1. Wash wells of overnight culture by removing well contents and pipetting in
| |
| - | 350 μl of double distilled water.
| |
| - | <br> 2. Aspirate immediately by touching pipette to the side of the plate.
| |
| - | <br> 3. Add 350 μL of CV stock solution to well.
| |
| - | <br> 4. Incubate for 30 min. at room temperature.
| |
| - | <br> 5. Aspirate all of the liquid.
| |
| - | <br> 6. Repeat 350 μl double distilled water wash 3 times. Make sure to empty the
| |
| - | well completely on the last wash.
| |
| - | <br> 7. Add 350 μL of 80:20 ethanol:acetone solution.
| |
| - | <br> 8. Incubate for 15 min. while gently shaking.
| |
| - | <br> 9. Transfer the entire amount to an empty well on the microtiter plate.
| |
| - | <br> 10.Measure absorbance at 600 nm.
| |
| - |
| |
| - | <br><br><b><u>Agglutination</u></b><br/>
| |
| - | <b>Principle:</b>Fimbriae bind mannose. Yeast cells display mannose molecules on their
| |
| - | cell surface and can be induced to visibly clump together with E. coli cells that
| |
| - | express functional fimbriae under the specific culture conditions.<br>
| |
| - | <b>Materials:</b>
| |
| - | <br> • Mortar and pestle
| |
| - | <br> • Prepare the stock solution (yeast slurry) fresh on the day of measurement:
| |
| - | <br> Add half a teaspoon of Fleischmann's yeast granules.
| |
| - | <br> Using a mortar, grind the yeast to powder form. Add 500mg of yeast powder in 5mL of 10% PBS.
| |
| - | <br> • 96-well plate with clear bottom
| |
| - | <br><b>Procedure:</b>
| |
| - | <br> 1. Add 60 μL of the yeast slurry with 300 μL of overnight culture into well.
| |
| - | <br> 2. Mix briefly using a micropipette.
| |
| - | <br> 3. Score agglutination after 5 minutes on a scale of "–" to "+++", based on
| |
| - | <a href= "http://www.sciencedirect.com/science/article/pii/S0022283603005916">http://www.sciencedirect.com/science/article/pii/S0022283603005916</a>
| |
| - | <br></p>
| |
| - | <p style = "font-size:15px;">Nishiyama M., Vetsch M., Puorger C., Jelesarov I., Glockshuber R.
| |
| - | 2003. Identification and characterization of the chaperone-subunit
| |
| - | complex-binding domain from the type 1 pilus assembly platform FimD. J.
| |
| - | <i>Mol. Biol.</i> 330, 513–525.</p>
| |
| - | <img src="https://static.igem.org/mediawiki/2013/b/bd/Alaskdjflkasdf%3B.jpg" style="opacity: 1.0;important!">
| |
| - |
| |
| - |
| |
| - | <br><p style = "font-size:18px;"><b><u>Adhesion</u></b><br/>
| |
| - | <b>Principle:</b>To measure the number of adherent cells by washing microtiter wells and
| |
| - | determining remaining cells through increase of ethidium bromide (EtBr) fluorescence.<br>
| |
| - | <b>Materials</b>
| |
| - | <br> • EtBr stock solution: 225 μL of (0.5 g/L ethidium bromide in water), 75mg of
| |
| - | NaN<sub>3</sub>, and 15 mL of 1X TBE. Store in cool and dark place till usage.
| |
| - | <br> • 1:1000 dilution of Sigma Antifoam A
| |
| - | <br> • 96-well plate with clear bottom
| |
| - | <br><b>Procedure:</b>
| |
| - | <br> 1. Aspirate overnight culture volumes from wells.
| |
| - | <br> 2. Add 100 μl of EtBr stock solution.
| |
| - | <br> 3. Measure fluorescence with <tt>λ</tt><sup>ex</sup> 510 nm / <tt>λ</tt><sup>em</sup> 595 nm.
| |
| - |
| |
| - | <br><br><b><u>Colanic acid <a href="http://bio.huji.ac.il/upload/94(1).pdf">[http://bio.huji.ac.il/upload/94(1).pdf]</a></u></b><br/>
| |
| - | <b>Citation:</b> Ionescu, M., Belkin, S. (2009). Simple quantification of bacterial
| |
| - | envelope-associated extracellular materials, J. Microbiol.Methods,
| |
| - | doi:10.1016/j.mimet.2009.06.020
| |
| - | <br><b>Principle:</b> When evaluated against OD<sup>600</sup> or cell count, measuring the height of
| |
| - | each PCV tube provides fast and simple evaluation of small differences in
| |
| - | extracellular content.
| |
| - | <br><b>Materials:</b>
| |
| - | <br> • TPP easy read cell counter tubes (PCV tubes) from
| |
| - | <a href= "http://www.sigmaaldrich.com/catalog/product/sigma/z761001?
| |
| - | lang=en®ion=CA">http://www.sigmaaldrich.com/catalog/product/sigma/z761001?lang=en®ion=CA</a>
| |
| - | <br> • Microcentrifuge
| |
| - | <br><b>Procedure:</b>
| |
| - | <br> 1. Add 455 μL of cell culture into a clean PCV tube. Parafilm the opening.
| |
| - | <br> 2. Spin at 4,000 RCF for 2 min.
| |
| - | <br> 3. Note length of the packed cell column according to the graduations of the
| |
| - | capillary.
| |
| - | <br> 4. Packed cell volume is expressed as a value per OD<sup>600</sup>.
| |
| - |
| |
| - | <br><br><b><u>Colony Morphology</u></b><br/>
| |
| - | <b>Reasoning:</b>
| |
| - | <br>The dyes in the agar plates will differentially stain cells based on whether or not they produce
| |
| - | curli and cellulose (Nakhamchik 2008). Bacteria that exclusively express curli will bind to the
| |
| - | congo red and will form red colonies, whereas bacteria that express only cellulose will form blue
| |
| - | colonies. If both are expressed, purple colonies will be formed. Rough colonies are indicative of
| |
| - | curli formation. In the below photograph, there is more variation in colour at 23°C (picture
| |
| - | on the left) than at 37°C, reflecting the protein expression phenotypes that are accessible
| |
| - | at the lower temperature. This is the reason the former temperature was chosen to incubate
| |
| - | colony morphology plates.</p>
| |
| - |
| |
| - | <p style = "font-size:15px;"><br>Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved
| |
| - | between <i>Salmonella typhimurium</i> and <i>Escherichia coli</i> with Respect to Operon Structure and
| |
| - | Regulation.<i>J. Bacteriol</i>,vol. 180 no. 3,722-731.</p>
| |
| - | <p style = "font-size:18px;">
| |
| - |
| |
| - | <img src= "https://static.igem.org/mediawiki/2013/1/16/Aksdfjlskdlwwkelqfn.png">
| |
| - | <b>Materials</b>
| |
| - | <br> • LB Agar with 40 µg/mL Congo Red dye and 20 µg/mL Brilliant Blue dye
| |
| - | <br> • Petri dishes
| |
| - | <br> • Antibiotic
| |
| - | <br> • Inoculation loop for streaking
| |
| - | <br><b>Protocol</b>
| |
| - | <br> 1. Prepare phosphate-buffered LB Agar containing 40 µg/mL Congo Red dye and 20 µg/mL
| |
| - | Brilliant Blue dye along with appropriate antibiotic.
| |
| - | <br> 2. Prepare overnight cultures of <i>E. coli</i> in phosphate buffered media.
| |
| - | <br> 3. To save plates, divide each plate into 4 quarters using a lab marker or use a whole plate.
| |
| - | Streak out bacteria from overnight culture using an inoculation loop.
| |
| - | <br> 4. Incubate in darkness at 23°C for 48 h.
| |
| - | <br> 5. Assess colony morphology.
| |
| - |
| |
| - |
| |
| - | <br><br><b><u>Congo Red</u></b><br/>
| |
| - | <b>Reasoning:</b><br>
| |
| - | CR binds to curli and cellulose. The difference between the blank CR absorbance and absorbance
| |
| - | of the supernatant taken after centrifugation will be proportional to the amount of CR by our
| |
| - | substrates of interest.</p>
| |
| - | <p style = "font-size:15px;">
| |
| - | Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved
| |
| - | between <i>Salmonella typhimurium</i> and <i>Escherichia coli</i> with Respect to Operon Structure and
| |
| - | Regulation.<i>J. Bacteriol</i>,vol. 180 no. 3,722-731.
| |
| - | <br>W. E. Klunk, R. F. Jacob, and R. P. Mason, “Quantifying amyloid by congo red spectral shift
| |
| - | assay,” <i>Methods in Enzymology</i>, vol. 309, pp. 285–305, 1999.</p>
| |
| - | <p style = "font-size:18px;">
| |
| - | <b>Materials for Congo Red:</b>
| |
| - | <br> • 0.5 mM CR solution in <sup>dd</sup>H<sup>2</sup>O
| |
| - | <br> • Antifoam A concentration
| |
| - | <br> • 96-well plate with clear bottom
| |
| - | <br> • Microcentrifuge tubes
| |
| - | <br> • Centrifuge
| |
| - | <br> • Absorbance plate reader
| |
| - | <br><b>Protocol</b>
| |
| - | <br> 1. To a 0.5 mM Congo red (CR) in <sup>dd</sup>H<sup>2</sup>O, add 15 μL of Antifoam A concentrate per 100
| |
| - | mL (final concentration 10ppm per well on 96-well plate) to prevent bubbles while
| |
| - | pipetting.
| |
| - | <br> 2. Pipette 200 μL of 1:100 dilution of overnight culture grown in phosphate buffered LB
| |
| - | into 96-well plate well.
| |
| - | <br> 3. Add 100 μL of phosphate buffered LB.
| |
| - | <br> 4. Incubate for 48 h at 23°C in darkness.
| |
| - | <br> 5. Add 20 μL of the 0.5 mM Congo red solution to well containing bacteria grown for 48 h.
| |
| - | <br> 6. Pipette 150 μL of CR into another well for a blank measurement (the “blank CR” well).
| |
| - | <br> 7. Incubate for 60 min.
| |
| - | <br> 8. Transfer 200 μL into microcentrifuge tube.
| |
| - | <br> 9. Centrifuge at 14,000 x g for 30 seconds.
| |
| - | <br> 10.Transfer 150 μL to an empty well on the microtiter plate.
| |
| - | <br> 11.Measure absorbance at 490 nm.
| |
| - | <br> 12.Subtract absorbance value from absorbance value of the“blank CR”. This will give you
| |
| - | the amount of bound CR.
| |
| - |
| |
| - |
| |
| - | <br><br><b><u>Calcofluor</u></b><br/>
| |
| - | <b>Reasoning:</b><br>
| |
| - | CF binds to cellulose. The difference between the blank CF fluorescence and fluorescence of the
| |
| - | supernatant taken after centrifugation will be proportional to the amount of CF bound by our
| |
| - | substrates of interest.
| |
| - | </p>
| |
| - |
| |
| - | <p style = "font-size:15px;"><br>Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved
| |
| - | between <i>Salmonella typhimurium</i> and <i>Escherichia coli</i> with Respect to Operon Structure and
| |
| - | Regulation.<i>J. Bacteriol</i>,vol. 180 no. 3,722-731.</p>
| |
| - | <p style = "font-size:18px;">
| |
| - | <b>Materials for Calcofluor:</b>
| |
| - | <br> • 0.02 g/L CF solution in <sup>dd</sup>H<sup>2</sup>O
| |
| - | <br> • Antifoam A concentration
| |
| - | <br> • Black 96-well plate
| |
| - | <br> • Microcentrifuge tubes
| |
| - | <br> • Centrifuge
| |
| - | <br> • Fluorescence plate reader
| |
| - | <br><b>Protocol</b>
| |
| - | <br> 1. The original calcofluor (CF) solution contained Evan's blue 0.5 g/L, CF 1.0 g/L in water
| |
| - | (as purchased from Sigma). To a working solution of 0.02 g/L CF of original solution, add
| |
| - | 15 μL of Antifoam A concentrate per 100 mL (final concentration 10ppm in each well of
| |
| - | 96-well plate).
| |
| - | <br> 2. Pipette 200 μL of 1:100 dilution of overnight culture grown in phosphate buffered LB
| |
| - | into 96-well plate well.
| |
| - | <br> 3. Add 100 μL of phosphate buffered LB.
| |
| - | <br> 4. Incubate for 48 h at 23 degrees C in darkness.
| |
| - | <br> 5. Add 20 μL of 0.02 g/L CF stock solution to first well.
| |
| - | <br> 6. Pipette 150 μL of CF into another well for a blank measurement (the “blank CF” well).
| |
| - | <br> 7. Incubate for 60 min.
| |
| - | <br> 8. Transfer 200 μL of first well contents into microcentrifuge tube.
| |
| - | <br> 9. Centrifuge at 14,000 x g for 30 seconds.
| |
| - | <br> 10.Transfer 150 μL to an empty well on the microtiter plate.
| |
| - | <br> 11.Measure fluorescence with <tt>λ</tt><sup>ex</sup> 352nm / <tt>λ</tt><sup>em</sup> 450 nm.
| |
| - | <br> 12.Subtract fluorescence value from fluorescence of “blank CF” well to get amount of
| |
| - | bound CF.
| |
| - |
| |
| - | </p>
| |
| - | </font>
| |
| - | </h7>
| |
| - | </div>
| |
| - |
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <br/>
| |
| - |
| |
| - |
| |
| - | </html>
| |
"
