Team:Toronto/Project/Data

DATA

Assay/ Stimuli
Each assay/stimuli needs a protocol, and materials list. We need to write out the protocol using both the stimuli inoculation template files as well as the assay files as our guidelines since these are the most updated procedures we're currently following. Plus, we also need to write out protocols for the Motility and Colony Morphology assays.

Aggregation
Principle: measure cell density in culture below surface, aggregated cells will have sunk to the bottom of the well.
Protocol:
1. Remove 155 μL of culture volume from the wells, being careful to neither aspirate cells from the surface of the culture nor from the bottom of the well.
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 μL).
3. Measure OD 600

Cell Density
Principle: OD 600 is used as a surrogate for number of cells.
    Note: we can't dilute culture volumes so actual cell numbers for higher ODs have to be calibrated.
Protocol:
1. Measure OD 600 for the entire plate in the microtiter plate reader. Use the crystal violet protocol (iGEM CV).

Crystal Violet
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.
Notes: Depletion of the stock solution should not be quantitative but leave room to detect increased amounts of binders.
Protocol:
Stock solution: 0.3 g/L crystal violet in water; ethanol/acetone 80:20 v:v
1. Aspirate remaining culture volumes from wells for which aggregation was measured
2. Wash wells by pipetting in 350μl of dd water, and reaspirate immediately by touching pipette to the side of the plate.
3. Add 350 μL of stock solution to well.
4. Incubate for 30 min. while gently shaking
5. Aspirate all of the liquid
6. Repeat dd water wash 3x being careful to empty the well completely on the last wash.
7. Add 350 μL of ethanol/acetone
8. Incubate for 15 min. while gently shaking
9. Transfer the entire amount to an empty well on the microtiter plate.
10.Measure absorbance at 600 nm with "iGEM CV" protocol.

Yeast Agglutination
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.
Protocol:
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).
1. Add 60 μL of the yeast suspension to a well.
2. Mix briefly by aspiration.
3. Score agglutination after 5 minutes on a scale of "–" to "+++". (Refer to Fig. 6A of the reference for comparison).
Reference - modified from:
http://www.ncbi.nlm.nih.gov/pubmed/12842468

Motility
Principle: Motile bacteria form diffuse colonies in low-concentration agar.
Protocol:
Plates: overlay 5mL of 0.3% agar on colony morphology agar plates (use sterile glass pipette).
1. Dip pipette tip into overnight culture.
2. Inoculate one dot on plate.3. Measure diameter of colony at 12h increments.
3. Measure diameter of colony at 12h increments.