Template:SBB- Streptavidin ELESA

NOTE: Binding of bacteria to the plate is tenuous and not reducible at this time. Different strains also act very differently. This assay is no longer being developed.

Enzyme-linked Ecoli-sorbent Assay

Positive control is pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx} (this also contains a strep tag and has been tested for display)

Another handy positive control for adhesion is a bacteria that expresses RFP (to view binding to the plate)

Negative control is pBca9495CA-Bca1363 {Pbad.rbs.prepro.StrepTag}

Wells containing ??? should be included to measure background.

NOTE: All washes should be done on the autowasher in Chris' lab.

Adhesion

1. Grow cells to saturation and induce with arabinose. Typically, inoculate overnight, add arabinose in the morning, and assay in the afternoon. Before assaying, measure OD600.

2. Prepare a solution of 20 ug/mL Ribonuclease B in 0.02M Sodium Bicarbonate.

3. Add 100uL of the RNaseB to each well of a 96-well TC-treated PS plate.

4. Incubate at 37C for 1 hr with no shaking.

5. Plate wash 3x with 100uL PBS.

6. Block by adding 100uL of 1.0 mg/mL BSA in PBS.

7. Incubate at 37C for 30 min with no shaking.

8. Aspirate out the block, then add the bacteria (5uL bacteria + 95uL of 1.0 mg/mL BSA in PBS gives decent coverage, less than ~5% of the plate area - we should add more next time)

9. Incubate at 37C for 40 min with no shaking.

10. Plate wash 3x with PBS. View in microscope to determine if cells are adhering to the surface of the plate.

Assay

To be written if and when the adhesion part is working.