This test will vary several circuit parameters to find the ideal sensor and characterize total sensor behavior.

This will determine what amounts of Syk-TEVp for each placement of TCS-Gal4VP16 produce the best on/off ratio.

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, beta amyloid (if beta amyloid fails, anti IgM can be used to force the receptor into an active state)

 

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

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.

 

We should identify what level of Syk-TEVp produces the best on/off ratio. 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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