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Description: 

In this group of experiments, we are aiming to test the affinity of our receptors, LilrB2 and PirB to beta-amyloid oligomers. To do that, we will have HEK293 cells express LilrB2 and PirB, with and without fluorescent protein fusions. We will then apply biotinylated beta-amyloid oligomers to the LilrB2 and PirB expressing cells. To test for binding, we are going to use the biotin-binding protein streptavidin covalently bonded to a fluorophore. 

Experiments Included: 

Description: 

Making HEK293 expressing LilrB2 and PirB on the cell membrane. Will test binding to receptors using the beta-amyloid oligomers and monomers in Experiment 4.

Plan:

  • Order LilrB2 and PirB gBlocks 
  • Simulate Golden Gate on Geneious and make sure it works 
  • Golden Gate gBlocks into ggDONR
  • Transform bacteria with Golden Gate product and plate 
  • Pick colonies and grow in liquid culture
  • Miniprep cells 
  • LR into pDEST

Status:

  • First and second set of liquid cultures didn't grow, possibly because:
    • Too much media
    • Plates were old, antibiotic possibly not working
  • First set of plates didn't have any blue colonies
  • Second set of plates grew blue and white colonies
    • PirB didn't grow any white colonies
    • Colonies picked for LilrB2
      • Liquid cultures grew
      • Miniprepped - Product to be verified
  • Golden Gate repeated
    • cells transformed and plated
    • +ve control didn't grow
      • LilrB2 grew blue and white colonies
      • PirB grew white colonies


Experiment 2: Making HEK293 cells expressing LilrB2 and PirB fused with fluorescent protein

Experiment 3: Oligomerizing beta-amyloid from beta-amyloid monomers 

Experiment 4: Testing binding of monomers and oligomers to the cells from Experiment 1 and Experiment 2

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