Difference between revisions of "Team:WPI-Worcester/Harvard-Collaboration"

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<h2>WPI and Harvard Biodesign Collaboration </h2>
 
<h2>WPI and Harvard Biodesign Collaboration </h2>
  
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<p><h3>Biofilm Assay Protocol</h3></p>
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<p><h3>WPI</h3></p>
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<p style="text-indent: 2em;"><h10>We completed the biofilm assay described above with strains of E. coli with modified pili created by the Harvard team. The strains we tested were JW4283, a fim H knockout and negative control for biofilm formation, and C2M+C5 and C9-3+C5, which express modified pili when induced by arabinose and rhamnose. Upon receiving agar stabs from the Harvard team, we streaked plates and incubated them at 37oC for 24 hours. Afterwards, we placed the plates in the refrigerator until we received the arabinose and rhamnose needed to induce pili formation in the experimental strains. Once we had all necessary materials, we grew 5mL liquid cultures of each of the 3 strains in LB in accordance with the protocol. The JW4283 culture was not supplemented with antibiotics or inducers, but the C2M+C5 and C9-3+C5 cultures were supplemented with 5 μL of chloramphenicol, 5 μL of ampicillin, 0.01% arabinose, and 0.5% rhamnose. We prepared the 1:100 dilutions in LB broth and M9 minimal media, which we found to support biofilm formation while designing our biofilm assay. The remainder of the protocol was left unchanged. Below are the results of the assay.</h10></p>
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</h10>Materials Needed:</p>
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</h10> 1 round-bottom 96-well plate</p>  
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</h10>1 flat-bottom 96-well plate</p>
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</h10>Appropriate media</p>
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</h10>Appropriate antibiotic stock</p>
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</h10>15 mL conical tubes or glass test tubes for growing liquid cultures</p>
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</h10>0.1% crystal violet</p>
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</h10>30% acetic acid</p>
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</h10>Paper towels</p>
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</h10>A large beaker</p>
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</h10>A tray or box that is slightly larger than a 96-well plate</p>
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</h10>A plate reader</p>
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<p></p>
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</h10>1. Prepare 5mL LB liquid cultures (supplemented with appropriate antibiotics and inducers) from stock or a colony from the transformation plate of the desired strains. Allow the cultures to grow for 18-20 hours at 37 degrees Celsius in a shaking incubator.</p>
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</h10>2. Prepare 1:100 dilutions, with a total volume of 1 mL, with desired media for each liquid culture.</p>
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</h10>3. Plate 100 μL of each dilution in sets of 4 wells in a round-bottom 96 well plate.</p>
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</h10>4. Cover the 96-well plate and incubate at 37oC for 48 hours.</p>
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</h10>5. Shake the plate out over a tray to remove all planktonic bacteria.</p>
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</h10>6. Rinse the 96-well plate in a large beaker of water and shake the water out over the tray.</p>
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</h10>7. Lay a paper towel out on the bench top. Hit the 96-well plate against the covered bench top until no liquid remains in the wells.</p>
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</h10>8. Stain all wells used in the assay with 125 μL of 0.1% crystal violet for 10 minutes.</p>
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</h10>9. Shake the 96-well plate over the tray again and rinse out the crystal violet in a large beaker of water.</p>
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</h10>10. Cover the bench top with more paper towels and hit the plate against the bench top until all wells are free of liquid crystal violet. Note: Make sure that the only crystal violet remaining is bound to a biofilm at the bottom of a well. Rings of crystal violet around a well are not indicative of biofilm formation and should be rinsed again, as excess stain will skew the results of the assay.</p>
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</h10>11. Leave the plate face up on the bench top overnight to dry.</p>
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</h10>12. Add 200 μL of 30% acetic acid to all wells that were stained to solubilize the crystal violet. Allow the acetic acid to sit for 10 minutes.</p>
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</h10>13. Pipette up and down the mix the acetic acid and crystal violet in the wells.<h10>
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</h10>14. Transfer 125 μL of the acetic acid/crystal violet solution from each well into a well in a flat-bottom 96-well plate.</p>
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</h10>15. Read the OD595nm of each well in the flat-bottom plate with a plate reader.</p>
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</h10>16. Subtract the average of the blank wells from the OD of each well that contained a sample.</p>
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</h10>17. Calculate the average of the sets of wells containing the same sample.</p>
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</h10>18. Normalize the averages to the average of the control wells.</p>
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Revision as of 18:57, 18 September 2015


WPI and Harvard Biodesign Collaboration

WPI

We completed the biofilm assay described above with strains of E. coli with modified pili created by the Harvard team. The strains we tested were JW4283, a fim H knockout and negative control for biofilm formation, and C2M+C5 and C9-3+C5, which express modified pili when induced by arabinose and rhamnose. Upon receiving agar stabs from the Harvard team, we streaked plates and incubated them at 37oC for 24 hours. Afterwards, we placed the plates in the refrigerator until we received the arabinose and rhamnose needed to induce pili formation in the experimental strains. Once we had all necessary materials, we grew 5mL liquid cultures of each of the 3 strains in LB in accordance with the protocol. The JW4283 culture was not supplemented with antibiotics or inducers, but the C2M+C5 and C9-3+C5 cultures were supplemented with 5 μL of chloramphenicol, 5 μL of ampicillin, 0.01% arabinose, and 0.5% rhamnose. We prepared the 1:100 dilutions in LB broth and M9 minimal media, which we found to support biofilm formation while designing our biofilm assay. The remainder of the protocol was left unchanged. Below are the results of the assay.

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