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Description: 

In this group of experiments, we are aiming to test whether or not our receptor proteins localize to the cell membrane. To do that, we will have HEK293 cells express LilrB2 and PirB. We will then use anti-LilrB2 and anti-PirB antibodies to test for membrane localization. 

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 membrane localization by using anti-LilrB2 and anti-PirB antibodies in Experiment 2.

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
    • Both liquid cultures grew


Experiment 2: Testing membrane localization of LilrB2 and PirB

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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