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| | <h7><font color=black><p style = "text-align:center; font-size:35px;"><b>DATA</b></p><br/> | | <h7><font color=black><p style = "text-align:center; font-size:35px;"><b>DATA</b></p><br/> |
| - | <p style = "font-size:18px;"><b><u>Assay/ Stimuli</u></b><br/> | + | <p style = "font-size:18px;"> |
| - | Each assay/stimuli needs a protocol, and materials list. We need to write out
| + | The following graphs show our relative spectrophotometer measurements for |
| - | the protocol using both the stimuli inoculation template files as well as the
| + | each assay axis when paired with each stimuli, for a specific strain. The 2 |
| - | assay files as our guidelines since these are the most updated procedures
| + | controls are wild type MG1655 and BW25113. The other strains tested have |
| - | we're currently following. Plus, we also need to write out protocols for the
| + | been transformed with pEBS and induced with 0.3mM IPTG. <br/> |
| - | Motility and Colony Morphology assays.<br/>
| + | <b>BW: BW25113</b><br/> |
| - | <p style = "font-size:18px;"><b><u>Aggregation</u></b><br/>
| + | <b>MG: MG1655</b><br/> |
| - | Principle: measure cell density in culture below surface, aggregated
| + | <b>BWC: BW25113 WITH csgD</b><br/> |
| - | cells will have sunk to the bottom of the well.<br/>
| + | <b>BWF: BW25113 WITH fimB</b><br/> |
| - | Protocol:<br/>
| + | <b>BWM: BW25113 WITH mlrA</b><br/> |
| - | 1. Remove 155<tt> μ</tt>L of culture volume from the wells, being careful to
| + | <b>BWO: BW25113 WITH ompA</b><br/> |
| - | neither aspirate cells from the surface of the culture nor from the
| + | <b>BWY: BW25113 WITH csgD</b><br/> |
| - | bottom of the well.<br/>
| + | |
| - | 2. Place into empty microtiter well (at the edge of the plate) but end the
| + | |
| - | pipetting at the first stop so no bubble gets into the well (we assume
| + | |
| - | that this is 150 <tt>μ</tt>L).</br>
| + | |
| - | 3. Measure OD 600</br>
| + | |
| - | | + | |
| - | <p style = "font-size:18px;"><b><u>Cell Density</u></b><br/>
| + | |
| - | Principle: OD 600 is used as a surrogate for number of cells.</br>
| + | |
| - | Note: we can't dilute culture volumes so actual cell numbers for higher
| + | |
| - | ODs have to be calibrated.</br>
| + | |
| - | Protocol:</br>
| + | |
| - | 1. Measure OD 600 for the entire plate in the microtiter plate reader. Use
| + | |
| - | the crystal violet protocol (iGEM CV).</br>
| + | |
| - | <p style = "font-size:18px;"><b><u>Crystal Violet</u></b><br/>
| + | |
| - | Principle: Crystal violet binds to polysaccharides of the biofilm matrix.
| + | |
| - | Since we are interested in the amount of biofilm that is produced, we
| + | |
| - | measure binding to adhering biofilm (if any exists). A stock solution of
| + | |
| - | crystal violet is incubated with the culture, the supernatant is removed,
| + | |
| - | bound crystal violet is mobilized with an ethanol/acetone solution and the
| + | |
| - | absorption is determined.</br>
| + | |
| - | Notes: Depletion of the stock solution should not be quantitative but
| + | |
| - | leave room to detect increased amounts of binders.</br>
| + | |
| - | Protocol:</br>
| + | |
| - | Stock solution: 0.3 g/L crystal violet in water; ethanol/acetone
| + | |
| - | 80:20 v:v</br>
| + | |
| - | 1. Aspirate remaining culture volumes from wells for which aggregation
| + | |
| - | was measured</br>
| + | |
| - | 2. Wash wells by pipetting in 350<tt>μ</tt>l of dd water, and reaspirate
| + | |
| - | immediately by touching pipette to the side of the plate.</br>
| + | |
| - | 3. Add 350 <tt>μ</tt>L of stock solution to well.</br>
| + | |
| - | 4. Incubate for 30 min. while gently shaking</br>
| + | |
| - | 5. Aspirate all of the liquid</br>
| + | |
| - | 6. Repeat dd water wash 3x being careful to empty the well completely
| + | |
| - | on the last wash.</br>
| + | |
| - | 7. Add 350 <tt>μ</tt>L of ethanol/acetone</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 with "iGEM CV" protocol.</br>
| + | |
| - | | + | |
| - | | + | |
| - | <p style = "font-size:18px;"><b><u>Yeast Agglutination</u></b><br/>
| + | |
| - | Principle: 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>
| + | |
| - | Protocol:</br>
| + | |
| - | Stock solution: (freshly prepared on the day of measurement)
| + | |
| - | Half a teaspoon of Fleischmann's yeast granules are ground to a
| + | |
| - | powder in a mortar. A slurry of 500mg in 5ml PBS is prepared (10%
| + | |
| - | w/v).</br>
| + | |
| - | 1. Add 60 μL of the yeast suspension to a well.</br>
| + | |
| - | 2. Mix briefly by aspiration.</br>
| + | |
| - | 3. Score agglutination after 5 minutes on a scale of "–" to "+++". (Refer
| + | |
| - | to Fig. 6A of the reference for comparison).</br>
| + | |
| - | Reference - modified from: </br>
| + | |
| - | <a href="http://www.ncbi.nlm.nih.gov/pubmed/12842468">http://www.ncbi.nlm.nih.gov/pubmed/12842468</a>
| + | |
| - | | + | |
| - | <p style = "font-size:18px;"><b><u>Motility</u></b><br/>
| + | |
| - | Principle: Motile bacteria form diffuse colonies in low-concentration
| + | |
| - | agar.</br>
| + | |
| - | Protocol:</br>
| + | |
| - | Plates: overlay 5mL of 0.3% agar on colony morphology agar
| + | |
| - | plates (use sterile glass pipette).</br>
| + | |
| - | 1. Dip pipette tip into overnight culture.</br>
| + | |
| - | 2. Inoculate one dot on plate.3. Measure diameter of colony at 12h increments.</br>
| + | |
| - | 3. Measure diameter of colony at 12h increments.</br>
| + | |
| - | | + | |
| | | | |
| | | | |
| | + | See .pdf <a href="https://static.igem.org/mediawiki/2013/2/2d/Produced_by_convert-jpg-to-pdf.net.pdf">here</a> |
| | | | |
| | </p> | | </p> |
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