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Expand
titlePlate map

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

 
Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

 

 
Well 7

HEK293

Well 8

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 9

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

 

Well 10

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 11

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

 

 

Results:

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng





aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng



Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng


aBeta 500 nM

Strep 10 ug/mL

 

Image ModifiedImage ModifiedImage ModifiedImage ModifiedImage Modified
Image ModifiedImage ModifiedImage ModifiedImage ModifiedImage Modified
Expand
titleGating

1. BFP +

2.

Image Modified

 

BCR -

AB +

BCR +

AB +

OverlayImage Removed

Image Removed
 
 

7.15

 

#1

 
Image Added
 
Image Added

Discussion:

 

 

 

Expand
titleProgress
CloningTransfectionStainingCytometryAnalysisiGEM parts07/13/1507/15/1507/15/15
Image Added

7.22

#1

Image AddedImage AddedImage Added

7.22

#2

07/16/15

   

 

 

Discussion:

Expand
titleBackground

Testing Gantenerumab binding to beta amyloid oligomers in the context of the B-Cell Receptor. Repeat of experiment done during iGEM.

Expand
titleApproach

Testing beta amyloid binding to cells expressing the components of the B-Cell Receptor. To do this, we are going to treat cells with a saturating concentration of biotin-conjugated beta amyloid. Subsequently, we are going to treat with AlexaFluor 594 conjugated streptavidin. If beta-amyloid binds to the receptor, streptavidin can bind biotin and be detected via flow cytometry. We are controlling for eBFP bleed through into the red channel, background beta-amyloid binding and background streptavidin binding.

Expand
titleParts Needed

hEF1a:eBFP

hEF1a:GMabH

hEF1a:GmabL

hEF1a:CD79A

hEF1a:CD79B

 

 

Expand
titlePlate map

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

 
Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng





aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng



Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng


aBeta 500 nM

Strep 10 ug/mL

 

 
Well 7

HEK293

Well 8

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 9

HEK293

hEF1a:eBFP 100ng

Dummy 400ng





aBeta 500 nM

Strep 10 ug/mL

 

Well 10

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng



Strep 10 ug/mL

Well 11

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng


aBeta 500 nM

Strep 10 ug/mL

 

 

Results:

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

Well 1
HEK293
Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

 

Image RemovedImage RemovedImage RemovedImage RemovedImage RemovedImage RemovedImage RemovedImage RemovedImage RemovedImage Removed Expand
titleGating

1. BFP +

2.

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

 
Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

 

BCR -

AB +

BCR +

AB +

 

   

 

Image Removed

Discussion:

 

 

 

Expand
titleProgress
CloningTransfectionStainingCytometryAnalysis
iGEM parts07/13/1507/15/1507/15/1507/16/15
     
Expand
titleBackground

Testing Gantenerumab binding to beta amyloid oligomers in the context of the B-Cell Receptor. Repeat of experiment done during iGEM.

Expand
titleApproach

Testing beta amyloid binding to cells expressing the components of the B-Cell Receptor. To do this, we are going to treat cells with a saturating concentration of biotin-conjugated beta amyloid. Subsequently, we are going to treat with AlexaFluor 594 conjugated streptavidin. If beta-amyloid binds to the receptor, streptavidin can bind biotin and be detected via flow cytometry. We are controlling for eBFP bleed through into the red channel, background beta-amyloid binding and background streptavidin binding.

Expand
titleParts Needed

hEF1a:eBFP

hEF1a:GMabH

hEF1a:GmabL

hEF1a:CD79A

hEF1a:CD79B