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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 fused with YFP. 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. We will look for co-localization of the fluorophore and the beta-amyloid. 

Controls 

Add beta-amyloid to cells that are not expressing the Receptor-YFP fusion.

 

*We may potentially extend this experiment to trying to characterize the interaction between beta-amyloid and the receptors i.e. try to generate saturation plots and calculate dissociation constants.

 Cloning

 

Experiments Included:

AnchorB1B1Experiment B1

Description:

Make HEK293 expressing LilrB/PirB - YFP fusions. We will test binding beta-amyloid in Experiment B2

Plan:
Status:
  • PCRed extensions onto LilrB2 and PirB
    • gel run to verify PCR products 
      • LilrB2 did not give expected band size 
        • why? 
      • PirB did not give expected band size 
        • pENTR used as template was not right 
  • PCRed extensions onto YFP 
    • verification gel to be run 

 

Experiment B2

  •  Simulate PCR and Golden Gate on Geneious and make sure it PCR product will be compatible with eYFP and TCS-Gal4VP16 PCR products from other subgroups
  •  Golden Gate gBlocks into ggDONR
  •  Transform bacteria with Golden Gate product and plate 
  •  Pick colonies and grow in liquid culture
  •  Miniprep cells and verify product 
  •  Make glycerol stocks
  •  LR into pDEST
  •  Transform, pick colonies and grow in liquid culture
  •  Miniprep cells and verify product
  •  Make glycerol stocks 
  •  Midiprep product

 

Experiment

AnchorB2B2
Description:

Testing beta-amyloid binding to receptor proteins using biotinylated oligomerized beta-amyloid. We will test for binding using he biotin-binding protein streptavidin covalently bonded to a fluorophore. We will look for co-localization of the fluorophore and the beta-amyloid.

Plan:
  •  Oligomerize beta-amyloid monomers
Status:
Not yet started.  

 

Transfection Plan:

LilrB2


Expand
titleTrial 1
Expand
titleCytometry Plate
Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep


Well 4

HEK293

eBFP

LilrB2

Strep

Well 5

HEK293

eBFP

LilrB2

aBeta

Strep


Well 6
Well 7

 

Well 8

 

Well 9

 

Well 10

 

Well 11

 

Well 12
Well 13
Well 14
Well 15
Well 16
Well 17
Well 18
Well 19
Well 20
Well 21
Well 22
Well 23
Well 24
Expand
titleMicroscopy Plate

 

 

PirB

Expand
titleTrial 1
Expand
titleCytometry Plate
Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

PirB

Strep

Well 5

HEK293

eBFP

PirB

aBeta

Strep

 

Well 6
Well 7

 

Well 8

 

Well 9

 

Well 10

 

Well 11

 

Well 12
Well 13
Well 14
Well 15
Well 16
Well 17
Well 18
Well 19
Well 20
Well 21
Well 22
Well 23
Well 24
Expand
titleMicroscopy Plate

Live Microscopy

Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

PirB

Strep

Well 5

HEK293

eBFP

PirB

aBeta

Strep

 

Well 6
Well 1

HEK293

aBeta

Strep

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

PirB

Strep

Well 5

HEK293

eBFP

PirB

aBeta

Strep

 

Well 12

 

 

Expand
titleTrial 2
Expand
titleMicroscopy Plate

Fixed and stained

Well 1

HEK293

Well 2

HEK293

aBeta

Strep

Well 3

HEK293

eYFP

Dummy

Well 4

HEK293

eYFP

Dummy

aBeta

Strep

 

Well 5

HEK293

eYFP

PirB

Strep

Well 6

HEK293

eYFP

PirB

aBeta

Strep

 

Well 7

HEK293

 

Well 8

HEK293

aBeta

Strep


 

Well 9

HEK293

eYFP

Dummy

Well 10

HEK293

eYFP

Dummy

aBeta

Strep

 

Well 11

HEK293

eYFP

PirB

Strep

Well 12

HEK293

eYFP

PirB

aBeta

Strep

 

 

 

Results

LilrB2

Expand
titleLilrB2 Trial 1 Cytometry
Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

LilrB2

Strep

Well 5

HEK293

eBFP

LilrB2

aBeta

Strep

 

Image AddedImage AddedImage AddedImage AddedImage Added
 Image AddedImage AddedImage AddedImage Added
Expand
titleLilrB Microscopy Trial 1
 
Well 1

HEK293

Well 2

HEK293

aBeta

Strep

Well 3

HEK293

eYFP

Dummy

Well 4

HEK293

eYFP

Dummy

aBeta

Strep

 

Well 5

HEK293

eYFP

LilrB2

Strep

Well 6

HEK293

eYFP

LilrB2

aBeta

Strep


(a)  Image AddedImage AddedImage AddedImage Added
Untransfected stainedTransfected stained
Image AddedImage AddedImage Added
Image AddedImage AddedImage Added
Expand
titleLilrB2 Trial 2 Cytometry

Transfection marker: tagBFP

Staining: biotinylated beta amyloid and strep-AF594

Well 1

HEK293

(unstained)

Well 2

HEK293

(stained)

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

LilrB2

Strep

Well 5

HEK293

eBFP

LilrB2

aBeta

Strep

 

Image Added

Lots of background..

Image Added

Image AddedImage AddedImage Added
Duplicates
Image AddedImage AddedImage AddedImage AddedImage Added


PirB

Expand
titlePirB Cytometry Trial 1
Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

PirB

Strep

Well 5

HEK293

eBFP

PirB

aBeta

Strep

 

Image AddedImage AddedImage AddedImage AddedImage Added
 Image Added Image AddedImage Added
eBFP + Dummy; aBeta + StrepeBFP + PirB; aBeta + StrepOverlay
Image AddedImage AddedImage Added
MEAN: 171MEAN: 1069 
Expand
titlePirB Microscopy Trial 1
Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 4

HEK293

eBFP

PirB

Strep

Well 5

HEK293

eBFP

PirB

aBeta

Strep

 

Well 6
Well 7

HEK293

aBeta

Strep

Well 8

HEK293

eBFP

Dummy

Well 9

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 10

HEK293

eBFP

PirB

Strep

Well 11

HEK293

eBFP

PirB

aBeta

Strep

 

Well 12

Before staining

 Image AddedImage AddedImage AddedImage Added 
 Image AddedImage AddedImage AddedImage Added 

After staining

Image Added

sad cells after staining

(evos picture - blue channel)

 

eBFP and PirB (aBeta + Strep)

BlueRedComposite
Image AddedImage AddedImage Added
Image AddedImage AddedImage Added
Image AddedImage AddedImage Added

 

 

Expand
titlePirB Microscopy Trial 2
 
Well 1

HEK293

Well 2

HEK293

aBeta

Strep

Well 3

HEK293

eYFP

Dummy

Well 4

HEK293

eYFP

Dummy

aBeta

Strep

 

Well 5

HEK293

eYFP

PirB

Strep

Well 6

HEK293

eYFP

PirB

aBeta

Strep


(a)  Image AddedImage AddedImage AddedImage Added
(b)  Image AddedImage AddedImage AddedImage Added
Untransfected stainedTransfected stained
Image AddedImage AddedImage Added
Image AddedImage AddedImage Added

 

Analysis

LilrB2

Trial 1: 

The cell population transfected with LilrB2 does not show any increase in red fluorescence which may indicate that LilrB2 does not bind abeta. However, from the picture taken of the cells before staining, it appears that the transfection efficiency for that well is very low making the reliability of the results form this trial very questionable. 

PirB

Cytometry Trial 1: 

The cell population transfected with PirB shows a marked increase in red fluorescence which indicates that PirB is in fact binding abeta. Moreover, when the distribution of red fluorescence of the transfected population (gated using the blue fluorescence distribution) is plotted, you see a shift in the distribution towards more red fluorescence and the mean increases from 171 in the PirB - population to 1069 in the PirB + population. 

It is interesting to note the "non-linear" distribution of red vs. blue in the PirB + population. We do not begin to see a significant increase in red fluorescence until we reach the more highly transfected cells. It would be interesting to plot mean blue vs. mean red to try to analyze this further. 

  • Next steps: Microscopy! to try and confirm the results we are getting through cytometry