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titleParts Needed

BCR (CD79A+B (with and without TCS-Gal4VP16), IgM heavy, IgM light), Lyn, Syk-TEVp, tre-mKate(dox reporter circuit), UAS-eYFP(reporter circuit), hef1a:eBFP(transfection marker), (whatever is needed to make dox work).  We will be testing all combinations of CD79(A/B)-(stop/TCS-Gal4Vp16).

Dox

 

Plasmidfunction

pEXPR hEF1a: CD79A+STOP

test variable

pEXPR hEF1a: CD79A-TCS-Gal4VP16

test variable

pEXPR hEF1a: CD79B-TCS-Gal4VP16

test variable

pEXPR hEF1a: CD79B+STOP

test variable

pEXPR hEF1a: Gmab Heavy

BCR

pEXPR hEF1a: Gmab Light

BCR

pEXPR TRE: Syk-TEV

test variable

pEXPR hEF1a: Lyn+STOP

BCR
UAS:eYFPfinal reporter
TRE:mKatecorrelates to Syk-TEVp amount
hef1a:eBFPtransfection marker
Expand
titleSetup

Well 1

HEK293

Well 2

HEK293

 

Well 3

HEK293

 

Well 4

HEK293

 

Well 5 

HEK293

 

Well 6 

EMPTY

Well 7

HEK293

Well 8 

HEK293

 

Well 9 

HEK293

 

Well 10 

HEK293

 

Well 11 

HEK293

 

Well 12 

HEK293

Well 13 

HEK293

Well 14 

HEK293

Well 15 

HEK293

Well 16 

HEK293

Well 17 

HEK293 

Well 18 

HEK293

Well 19 

HEK293

Well 20

HEK293

Well 21 

HEK293

Well 22 

HEK293 

Well 23 

HEK293

 

Well 24 

HEK293

All combinations of plasmids being tested will be prepared and put into dox ladders

plasmids v  dox>011010010002000
CD79A-stop CD79B-stop      
CD79A-tcs-Gal4VP16 CD79B-stop      
CD79A-stop CD79B-tcs-Gal4VP1      
CD79A-tcs-Gal4VP1 CD79B-tcs-Gal4VP1      

First row is control for basal reporter expression.

 

Expand
titleExpected Results

We should identify what level of Syk-TEVp causes constitutive expression of the reporter and characterize the off behavior of our circuit so we will be able to compare it to the on behavior later. Our data will produce a scatter plot of eYFP (reporter) as a function of mKate (driven by the same promoter as syk).

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