Template:SBB- Flocculation Assay

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Flocculation Assay Protocol

To test cell-cell adhesion in the context of formation of flocculation (large clumps of boogers.)

Negative controls:

  • DH10B (must be the same strain as the one you are testing)

plasmid 1363 (a plasmid that infers antibiotic resistance with no displayer. 1363 is strep binding peptide in the periplasm)

Positive Control

  • DH10B (must be the same strain as the one you are testing)

with CPG-L6 displayer with a leucine zipper homo dimer part<GS5-IILK>

Protocol 1. Grow all of the parts to test along with the negative and positive controls in 96 well blocks with 2YT (media may need to change depending on the strain. For DH10B 2YT works the best for this assay.)
2. Add 400 ul of media to all wells and pick your samples from a plate. Do not induce. Grow overnight.
3. a. For every sample, take a culture tube and add 3ml of 2YT media and add 1 ul of each sample.
Note that for hetero dimers, add 1 ul of each hetero dimer to a single culture tube. Also, for a control (to test for homodimerization and aggregation) add 1ul of each heterodimer separately to 2 different 3ml tubes.
4. Check for large obvious clumps the next day.

Below are images of a clumping and no clumping. Note that in clumping, the supernatent is very, very clear.