This test will vary several circuit parameters to find the ideal sensor.

This will determine what amounts of Syk-TEVp for each placement of TCS-Gal4VP16 cause excessive constitutive production of reporter so we don't need to test those for the active test.  This will also characterize our circuits off behavior so we can compare it to its on behavior later to get the on/off ratio.  There is a test of no Gal4VP16 which will tell us how much constitutive eYFP our promoter produces.

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

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

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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